Smooth Muscle Growth Research Articles

Arsenic trioxide induces growth inhibition and death in human pulmonary artery smooth muscle cells accompanied by mitochondrial O2•- increase and GSH depletion.

Arsenic trioxide (ATO; As2 O3 ) induces cell death in various cells via oxidative stress. Expose to chronic arsenic is involved in the development of vascular diseases. However, little is known about the cytotoxic effects of ATO on human normal vascular smooth muscle cells (VSMCs). Thus, in this study, we investigated the effects of ATO on cell growth and death in human pulmonary artery smooth muscle (HPASM) cells in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. ATO treatment decreased the growth of HPASM cells with an IC50 of ∼30-50 μM at 24 h, and ATO induced HPASM cell death via apoptosis or necrosis dependent on the doses of it at this time. Treatment with 50 μM ATO did not increase ROS levels at the early time points, but it significantly increased mitochondrial O2•- levels at 24 h. ATO also induced GSH depletion in HPASM cells. N-acetyl cysteine (NAC; a well-known antioxidant) did not significantly affect apoptotic cell death, ROS levels, or GSH depletion in ATO-treated HPASM cells. However, l-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) intensified mitochondrial O2•- levels in ATO-treated HPASM cells, and significantly increased cell death and GSH depletion in these cells as well. In summary, we provided the first evidence that ATO inhibited the growth of HPASM cells, and induced apoptotic and/or necrotic cell death in these cells, accompanied by increases in mitochondrial O2•- level and GSH depletion.

Bradford C. Berk: Picking Good Questions.

As Brad Berk sees it, “All the work I do can pretty much fit under the heading of signal transduction in blood vessels.” This deceptively short phrase, however, covers decades of research into the cellular and genetic mechanisms that produce some of cardiovascular medicine’s most stubborn challenges: atherosclerosis, aneurysms, and pulmonary hypertension. Berk’s academic career has led him from the University of Rochester to Harvard Medical School, Emory University, and the University of Washington before circling back to Rochester. Currently, he holds the post of Distinguished University Professor in medicine, neurology, pathology, and pharmacology and physiology. He has founded and directed 2 multidepartment institutes: the Aab Cardiovascular Research Institute (CVRI; from 1999 to 2006) and the University of Rochester Neurorestoration Institute (URNI; from 2015 to the present). Bradford C. Berk While at the University of Washington, in 1998, he published with Oren Traub a review1 of what was then known about how changes in blood-flow profile (steady versus disturbed) influence vascular disease. Berk went on to develop a highly reproducible model2 for vascular remodeling of the carotid as mediated by blood-flow profile. The disturbed flow region shows the formation of intima—a pathological thickening of the vessel wall. Because this thickening is predictive for cardiovascular events, the identification of genetic factors that played a role in the process3 marked a significant advance in cardiovascular research. Together with Korshunov et al, Berk studied 3 disease processes that contribute to intima: endothelial dysfunction, inflammation, and smooth muscle growth. By performing a genetic linkage study to home in on regions of the mouse genome that contribute to the intima,4 Berk and Korshunov identified a specific gene ( RpL17 ) that accounts for smooth muscle cell growth. With Smolock et al,5,6 they found a key link to inflammation now identified …

Chronic IL-33 expression predisposes to virus-induced asthma exacerbations by increasing type 2 inflammation and dampening antiviral immunity

Rhinovirus infection triggers acute asthma exacerbations. IL-33 is an instructive cytokine of type 2 inflammation whose expression is associated with viral load during experimental rhinovirus infection of asthmatic patients. We sought to determine whether anti-IL-33 therapy is effective during disease progression, established disease, or viral exacerbation using a preclinical model of chronic asthma and invitro human primary airway epithelial cells (AECs). Mice were exposed to pneumonia virus of mice and cockroach extract in early and later life and then challenged with rhinovirus to model disease onset, progression, and chronicity. Interventions included anti-IL-33 or dexamethasone at various stages of disease. AECs were obtained from asthmatic patients and healthy subjects and treated with anti-IL-33 after rhinovirus infection. Anti-IL-33 decreased type 2 inflammation in all phases of disease; however, the ability to prevent airway smooth muscle growth was lost after the progression phase. After the chronic phase, IL-33 levels were persistently high, and rhinovirus challenge exacerbated the type 2 inflammatory response. Treatment with anti-IL-33 or dexamethasone diminished exacerbation severity, and anti-IL-33, but not dexamethasone, promoted antiviral interferon expression and decreased viral load. Rhinovirus replication was higher and IFN-λ levels were lower in AECs from asthmatic patients compared with those from healthy subjects. Anti-IL-33 decreased rhinovirus replication and increased IFN-λ levels at the gene and protein levels. Anti-IL-33 or dexamethasone suppressed the magnitude of type 2 inflammation during a rhinovirus-induced acute exacerbation; however, only anti-IL-33 boosted antiviral immunity and decreased viral replication. The latter phenotype was replicated in rhinovirus-infected human AECs, suggesting that anti-IL-33 therapy has the additional benefit of enhancing host defense.

ITE inhibits growth of human pulmonary artery endothelial cells

ABSTRACTAim: Pulmonary arterial hypertension (PAH), a deadly disorder is associated with excessive growth of human pulmonary artery endothelial (HPAECs) and smooth muscle (HPASMCs) cells. Current therapies primarily aim at promoting vasodilation, which only ameliorates clinical symptoms without a cure. 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an endogenous aryl hydrocarbon receptor (AhR) ligand, and mediates many cellular function including cell growth. However, the roles of ITE in human lung endothelial cells remain elusive. Herein, we tested a hypothesis that ITE inhibits growth of human pulmonary artery endothelial cells via AhR. Materials and Methods: Immunohistochemistry was performed to localize AhR expression in human lung tissues. The crystal violet method and MTT assay were used to determine ITE's effects on growth of HPAECs. The AhR activation in HPAECs was confirmed using Western blotting and RT-qPCR. The role of AhR in ITE-affected proliferation of HPAECs was assessed using siRNA knockdown method followed by the crystal violet method. Results: Immunohistochemistry revealed that AhR was present in human lung tissues, primarily in endothelial and smooth muscle cells of pulmonary veins and arteries, as well as in bronchial and alveolar sac epithelia. We also found that ITE dose- and time-dependently inhibited proliferation of HPAECs with a maximum inhibition of 83% at 20 µM after 6 days of treatment. ITE rapidly decreased AhR protein levels, while it increased mRNA levels of cytochrome P450 (CYP), family 1, member A1 (CYP1A1) and B1 (CYP1B1), indicating activation of the AhR/CYP1A1 and AhR/CYP1B1 pathways in HPAECs. The AhR siRNA significantly suppressed AhR protein expression, whereas it did not significantly alter ITE-inhibited growth of HPAECs. Conclusions: ITE suppresses growth of HPAECs independent of AhR, suggesting that ITE may play an important role in preventing excessive growth of lung endothelial cells.

Metalloproteinases in atherosclerosis

Atherosclerosis underlies most cardiovascular diseases, and is accepted as a primary cause of mortality worldwide. Proteases have been implicated in the development and progression of atherosclerosis, due to their ability to provoke focal destruction of the vascular extracellular matrix. Members of the metalloproteinase family, especially matrix metalloproteinases (MMPs), and their endogenous tissue inhibitors (TIMPs) have been suggested to perform complex dual roles during late-stage progression and rupture of atherosclerotic plaques. Proposed favourable actions of metalloproteinases include the promotion of vascular smooth muscle growth and survival which stabilises plaques, while conversely extracellular matrix destruction alongside interminable monocyte/macrophage accumulation can encourage plaque rupture. This review provides a summary of the cogent evidence connecting the contribution of individual metalloproteinases to atherosclerotic plaque development, progression, and instability. Topics discussed include structural, functional and cell-specific diversity of MMP members; evidence from animal models of atherosclerosis and comparisons with findings in humans; the dual role of MMPs and the requirement to selectively target individual MMPs; and the need for efficient surrogate markers of MMP inhibition. Accordingly, as our knowledge of the complex roles individual MMPs play especially during the progression and rupture of atherosclerotic plaques expands, new impetus is required for clinical trials evaluating the therapeutic potential of selective MMP inhibition, which could limit cardiovascular morbidity and mortality worldwide.

The Dynamic Actin Cytoskeleton in Smooth Muscle.

Smooth muscle contraction requires both myosin activation and actin cytoskeletal remodeling. Actin cytoskeletal reorganization facilitates smooth muscle contraction by promoting force transmission between the contractile unit and the extracellular matrix (ECM), and by enhancing intercellular mechanical transduction. Myosin may be viewed to serve as an "engine" for smooth muscle contraction whereas the actin cytoskeleton may function as a "transmission system" in smooth muscle. The actin cytoskeleton in smooth muscle also undergoes restructuring upon activation with growth factors or the ECM, which controls smooth muscle cell proliferation and migration. Abnormal smooth muscle contraction, cell proliferation, and motility contribute to the development of vascular and pulmonary diseases. A number of actin-regulatory proteins including protein kinases have been discovered to orchestrate actin dynamics in smooth muscle. In particular, Abelson tyrosine kinase (c-Abl) is an important molecule that controls actin dynamics, contraction, growth, and motility in smooth muscle. Moreover, c-Abl coordinates the regulation of blood pressure and contributes to the pathogenesis of airway hyperresponsiveness and vascular/airway remodeling in vivo. Thus, c-Abl may be a novel pharmacological target for the development of new therapy to treat smooth muscle diseases such as hypertension and asthma.

The effects of a growth hormone-releasing hormone antagonist and a gastrin-releasing peptide antagonist on intimal hyperplasia of the carotid artery after balloon injury in a diabetic rat model

IntroductionArterial restenosis after angioplasty/stenting has hindered coronary artery disease treatment, especially in diabetics. We theorized that gastrin-releasing peptide (GRP) antagonists and growth hormone-releasing hormone (GHRH) antagonists might decrease neointimal hyperplasia and restenosis in diabetic rats after common carotid arterial balloon injury. MethodsTwo separate experiments were conducted to test the effects of a GRP antagonist (RC-3095) and a GHRH antagonist (MZ-4-71) on vascular smooth muscle (VSM) growth. In a preliminary in vitro experiment non-injured human aortic vascular smooth muscle (VSM) proliferation was compared between growth media and control. In a second in vivo experiment, intimal and medial area, intima/media ratio (IM) and percent stenosis were compared between injured carotid arteries in twelve Zucker type II obese rats treated with subcutaneously injected RC-3095, MZ-4-71, or control media. ResultsIn the in vitro experiment, decreased VSM cell growth was observed in GRP antagonist (p < 0.05) and GHRH antagonist groups (p < 0.05) compared to the control group. In the in vivo experiment, the GRP antagonist group had a decreased IM ratio (1.63 ± 0.41, p < 0.05) and an increased area of stenosis (98.78% ± 1.48 p = NS) compared to control (2.38 ± 1.09) while the GHRH antagonist group had decreased IM ratio (1.33 ± 0.58 SD, p < 0.05) and percent area of stenosis (78.84% ± 24.97, p < 0.05) compared to control (2.38 ± 1.09). ConclusionsThe significant decrease in both IM ratio and percent area of stenosis in the GHRH antagonist group supports the hypothesis that this peptide may reduce neointimal hyperplasia and restenosis.

Matrix Metalloproteinase-1 Activation Contributes to Airway Smooth Muscle Growth and Asthma Severity.

Matrix metalloproteinase-1 (MMP-1) and mast cells are present in the airways of people with asthma. To investigate whether MMP-1 could be activated by mast cells and increase asthma severity. Patients with stable asthma and healthy control subjects underwent spirometry, methacholine challenge, and bronchoscopy, and their airway smooth muscle cells were grown in culture. A second asthma group and control subjects had symptom scores, spirometry, and bronchoalveolar lavage before and after rhinovirus-induced asthma exacerbations. Extracellular matrix was prepared from decellularized airway smooth muscle cultures. MMP-1 protein and activity were assessed. Airway smooth muscle cells generated pro-MMP-1, which was proteolytically activated by mast cell tryptase. Airway smooth muscle treated with activated mast cell supernatants produced extracellular matrix, which enhanced subsequent airway smooth muscle growth by 1.5-fold (P < 0.05), which was dependent on MMP-1 activation. In asthma, airway pro-MMP-1 was 5.4-fold higher than control subjects (P = 0.002). Mast cell numbers were associated with airway smooth muscle proliferation and MMP-1 protein associated with bronchial hyperresponsiveness. During exacerbations, MMP-1 activity increased and was associated with fall in FEV1 and worsening asthma symptoms. MMP-1 is activated by mast cell tryptase resulting in a proproliferative extracellular matrix. In asthma, mast cells are associated with airway smooth muscle growth, MMP-1 levels are associated with bronchial hyperresponsiveness, and MMP-1 activation are associated with exacerbation severity. Our findings suggest that airway smooth muscle/mast cell interactions contribute to asthma severity by transiently increasing MMP activation, airway smooth muscle growth, and airway responsiveness.

Morphological and growth responses of vascular smooth muscle and endothelial cells cultured on immobilized heparin and dextran sulfate surfaces.

Heparin has shown promise as a component of various biomaterial formulations, but its variable properties and inhibitory effects on some cell types have raised interest in use of dextran sulfate as an alternative. In this study, we characterized the interactions of vascular smooth muscle (SMC) and endothelial cells (EC) with heparin and dextran sulfate immobilized onto chitosan-based films. Films were modified by blending chitosan with type I collagen and covalently attaching heparin or dextran sulfate at various levels. Cell-material interactions were evaluated by quantifying cell spreading, shape and proliferation rate. ECs proliferated well on chitosan, but the polymer was a mediocre substrate for SMC growth. Immobilizing heparin on chitosan further inhibited SMC proliferation. However, blending collagen reversed the heparin inhibition of SMC growth, resulting in a pro-proliferative effect of heparin immobilized on chitosan-collagen films. Dextran sulfate surfaces supported both SMC and EC proliferation with or without the presence of collagen. The results indicate that inhibitory effects of heparin on SMC are reversed by immobilization in the presence of collagen, and that dextran sulfate may be superior to heparin as a biomaterial additive for promoting vascular cell growth in chitosan-based scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1725-1735, 2017.

Endothelial and smooth muscle cells derived from human cardiac explants demonstrate angiogenic potential and suitable for design of cell-containing vascular grafts

BackgroundEndothelial and smooth muscle cells are considered promising resources for regenerative medicine and cell replacement therapy. It has been shown that both types of cells are heterogeneous depending on the type of vessels and organs in which they are located. Therefore, isolation of endothelial and smooth muscle cells from tissues relevant to the area of research is necessary for the adequate study of specific pathologies. However, sources of specialized human endothelial and smooth muscle cells are limited, and the search for new sources is still relevant. The main goal of our study is to demonstrate that functional endothelial and smooth muscle cells can be obtained from an available source—post-surgically discarded cardiac tissue from the right atrial appendage and right ventricular myocardium.MethodsHeterogeneous primary cell cultures were enzymatically isolated from cardiac explants and then grown in specific endothelial and smooth muscle growth media on collagen IV-coated surfaces. The population of endothelial cells was further enriched by immunomagnetic sorting for CD31, and the culture thus obtained was characterized by immunocytochemistry, ultrastructural analysis and in vitro functional tests. The angiogenic potency of the cells was examined by injecting them, along with Matrigel, into immunodeficient mice. Cells were also seeded on characterized polycaprolactone/chitosan membranes with subsequent analysis of cell proliferation and function.ResultsEndothelial cells isolated from cardiac explants expressed CD31, VE-cadherin and VEGFR2 and showed typical properties, namely, cytoplasmic Weibel-Palade bodies, metabolism of acetylated low-density lipoproteins, formation of capillary-like structures in Matrigel, and production of extracellular matrix and angiogenic cytokines. Isolated smooth muscle cells expressed extracellular matrix components as well as α-actin and myosin heavy chain. Vascular cells derived from cardiac explants demonstrated the ability to stimulate angiogenesis in vivo. Endothelial cells proliferated most effectively on membranes made of polycaprolactone and chitosan blended in a 25:75 ratio, neutralized by a mixture of alkaline and ethanol. Endothelial and smooth muscle cells retained their functional properties when seeded on the blended membranes.ConclusionsWe established endothelial and smooth muscle cell cultures from human right atrial appendage and right ventricle post-operative explants. The isolated cells revealed angiogenic potential and may be a promising source of patient-specific cells for regenerative medicine.

137 Superparamagnetic Iron Oxide Nanoparticle Targeting of Adipose Tissue-Derived Stem Cells in Diabetes-Associated Erectile Dysfunction

To explore the effect of Adipose tissue-derived stem cells (ADSCs) labelled with superparamagnetic iron oxide nanoparticles (SPIONs) on improving the erectile function of streptozocin (STZ)-induced diabetic rats with an external magnetic field. Ten-week-old male Sprague Dawley rats were injected either with STZ to induce diabetes or citrate buffer as controls. Rat ADSCs were harvested and labelled with SPIONs, and then transplanted into corpus cavernosum of STZ-induced diabetic rats with or without an external magnetic field. At 28 days after transplantation, all rats were sacrificed for tracking of transplanted cells. Erectile response was assessed by cavernous nerve stimulation. Smooth muscle, endothelium and vascular endothelial growth factor (VEGF) expression in corpora cavernosum were assessed using immunohistochemistry. In vitro studies revealed that stem cell properties were minimally affected by SPION labeling. With magnetic targeting, SPION-labelled ADSCs were visibly attracted toward the magnet field. In vivo experiments, magnetic targeting of ADSCs contributed to long-term cell retention in corpus cavernosum and improved the erectile function of diabetic rats compared with ADSC injection alone. ADSCs were identified in the corpus cavernosums of diabetic rats at 28 days after transplantation, and they were negative for vWF and a-SMA, indicating that ADSCs did not differentiate into endothelial or smooth muscle cells. VEGF expression in the corpus cavernosum was significantly increased in both ADSC-treated groups compared with the PBS-treated group. The underlying mechanism of recovery appears to the paracrine mechanism of transplanted ADSCs.

Smooth muscle contraction and growth of stromal cells in the human prostate are both inhibited by the Src family kinase inhibitors, AZM475271 and PP2.

In benign prostatic hyperplasia, increased prostate smooth muscle tone and prostate volume may contribute alone or together to urethral obstruction and voiding symptoms. Consequently, it is assumed there is a connection between smooth muscle tone and growth in the prostate, but any molecular basis for this is poorly understood. Here, we examined effects of Src family kinase (SFK) inhibitors on prostate contraction and growth of stromal cells. SFK inhibitors, AZM475271 and PP2, were applied to human prostate tissues to assess effects on smooth muscle contraction, and to cultured stromal (WPMY-1) and c-Src-deficient cells to examine effects on proliferation, actin organization and viability. SFKs were detected by real time PCR, western blot and immunofluorescence in human prostate tissues, some being located to smooth muscle cells. AZM475271 (10μM) and PP2 (10μM) inhibited SFK in prostate tissues and WPMY-1 cells. Both inhibitors reduced α1 -adrenoceptor-mediated and neurogenic contraction of prostate strips. This may result from cytoskeletal deorganization, which was observed in response to AZM475271 and PP2 in WPMY-1 cells by staining of actin filaments with phalloidin. This was paralleled by reduced proliferation of wildtype but not of c-Src-deficient cells; cytotoxicity was mainly observed at higher concentrations (>50μM). In human prostate, smooth muscle tone and growth are both controlled by an SFK-dependent process, which may explain their common role in bladder outlet obstruction. Targeting prostate smooth muscle tone and prostate growth simultaneously by a single compound may, in principal, be possible.

Selective targeting of CREB-binding protein/β-catenin inhibits growth of and extracellular matrix remodelling by airway smooth muscle.

Asthma is a heterogeneous chronic inflammatory disease, characterized by the development of structural changes (airway remodelling). β-catenin, a transcriptional co-activator, is fundamentally involved in airway smooth muscle growth and may be a potential target in the treatment of airway smooth muscle remodelling. We assessed the ability of small-molecule compounds that selectively target β-catenin breakdown or its interactions with transcriptional co-activators to inhibit airway smooth muscle remodelling in vitro and in vivo. ICG-001, a small-molecule compound that inhibits the β-catenin/CREB-binding protein (CBP) interaction, strongly and dose-dependently inhibited serum-induced smooth muscle growth and TGFβ1-induced production of extracellular matrix components in vitro. Inhibition of β-catenin/p300 interactions using IQ-1 or inhibition of tankyrase 1/2 using XAV-939 had considerably less effect. In a mouse model of allergic asthma, β-catenin expression in the smooth muscle layer was found to be unaltered in control versus ovalbumin-treated animals, a pattern that was found to be similar in smooth muscle within biopsies taken from asthmatic and non-asthmatic donors. However, β-catenin target gene expression was highly increased in response to ovalbumin; this effect was prevented by topical treatment with ICG-001. Interestingly, ICG-001 dose-dependently reduced airway smooth thickness after repeated ovalbumin challenge, but had no effect on the deposition of collagen around the airways, mucus secretion or eosinophil infiltration. Together, our findings highlight the importance of β-catenin/CBP signalling in the airways and suggest ICG-001 may be a new therapeutic approach to treat airway smooth muscle remodelling in asthma.

Potassium Channels in Regulation of Vascular Smooth Muscle Contraction and Growth.

Potassium channels importantly contribute to the regulation of vascular smooth muscle (VSM) contraction and growth. They are the dominant ion conductance of the VSM cell membrane and importantly determine and regulate membrane potential. Membrane potential, in turn, regulates the open-state probability of voltage-gated Ca2+ channels (VGCC), Ca2+ influx through VGCC, intracellular Ca2+, and VSM contraction. Membrane potential also affects release of Ca2+ from internal stores and the Ca2+ sensitivity of the contractile machinery such that K+ channels participate in all aspects of regulation of VSM contraction. Potassium channels also regulate proliferation of VSM cells through membrane potential-dependent and membrane potential-independent mechanisms. VSM cells express multiple isoforms of at least five classes of K+ channels that contribute to the regulation of contraction and cell proliferation (growth). This review will examine the structure, expression, and function of large conductance, Ca2+-activated K+ (BKCa) channels, intermediate-conductance Ca2+-activated K+ (KCa3.1) channels, multiple isoforms of voltage-gated K+ (KV) channels, ATP-sensitive K+ (KATP) channels, and inward-rectifier K+ (KIR) channels in both contractile and proliferating VSM cells.

Isolation, expansion and characterization of porcine urinary bladder smooth muscle cells for tissue engineering.

BackgroundA key requirements for therapy utilizing the tissue engineering methodologies is use of techniques which have the capability to yield a high number of cells, from small tissue biopsy in a relatively short time. Up to date there was no optimal methods of isolation and expansion of urinary bladder smooth muscle cells (UB-SMCs). The aim of this study was to compare isolation and expansion techniques of UB-SMCs to select the most repeatable and efficient one.MethodFive protocols of porcine UB- SMCs isolation including enzymatic and explant techniques and three expansion techniques were compared. Isolation effectiveness was evaluated using trypan blue assay. Cell phenotype was confirmed by immunofluorescence staining. Proliferation rate was analyzed using MTT and X- Celligence system. Cellular senescence was assessed measuring β-galactosidase activity.ResultsEnzymatic methods using collagenase with dispase (method I) or collagenase only (method III) allowed to isolate much larger number of cells than the methods using trypsin with collagenase (method II) and collagenase after digestion with trypsin (method IV). The success rate of establishment of primary culture was the highest when the isolated cells were cultured in the Smooth muscle Growth Medium-2 (SmGM-2). Expression of the smooth muscle markers- alpha smooth muscle actin and smoothelin was the highest for cells isolated by enzymatic method I and cultured in SmGM-2. There was no significant signs of cell senescence until the 8th passage.ConclusionThe most efficient method of establishment of porcine UB-SMCs culture is enzymatic digestion of urinary bladder tissue with collagenase and dispase and culture of isolated cells in SmGM-2. This method was up to 10 times more efficient than other methods used for isolation and culture of UB-SMCs. This is an easy and consistent method for obtaining high numbers of urinary bladder smooth muscle cells.

Aeroallergen-induced IL-33 predisposes to respiratory virus-induced asthma by dampening anti-viral immunity

Abstract Frequent viral lower respiratory infections in early-life are an independent risk factor for asthma onset. This risk, and the development of persistent asthma, is significantly greater in children who later become sensitized. Here, we sought to elucidate the pathogenic processes that underlie the synergistic interplay between allergen exposures and viral infections. Mice were inoculated with a Pneumovirus (Pneumonia virus of mice; PVM) and exposed to low-dose cockroach extract (CRE) in early- and later-life, and airway inflammation, remodeling and hyperreactivity assessed. Mice were treated with anti-IL-33 or apyrase to neutralize or block the release of IL-33. PVM infection or CRE exposure alone failed to induce disease whereas PVM/CRE co-exposure acted synergistically to induce the features of asthma. CRE exposure during virus infection in early-life induced a biphasic IL-33 response and impaired IFN-a/l production, which in turn increased epithelial viral burden, airway smooth muscle growth, and type-2 inflammation. These features were ameliorated when CRE-induced IL-33 release was blocked or neutralized, while substitution of CRE with exogenous IL-33 recapitulated the phenotype observed in PVM/CRE co-exposed mice. Mechanistically, IL-33 down-regulated viperin and IFN regulatory factor 7 gene expression, and rapidly degraded IRAK1 expression in pDC, leading to TLR7 hypo-responsiveness and impaired IFN-a production. We identify a hitherto unrecognized function of IL-33 as a potent suppressor of innate antiviral immunity, and demonstrate that IL-33 contributes significantly to the synergistic interplay between respiratory virus and allergen exposures in the onset and progression of asthma.

Aeroallergen-induced IL-33 predisposes to respiratory virus–induced asthma by dampening antiviral immunity

Frequent viral lower respiratory infections in early life are an independent risk factor for asthma onset. This risk and the development of persistent asthma are significantly greater in children who later become sensitized. We sought to elucidate the pathogenic processes that underlie the synergistic interplay between allergen exposures and viral infections. Mice were inoculated with a murine-specific Pneumovirus species (pneumonia virus of mice [PVM]) and exposed to low-dose cockroach extract (CRE) in early and later life, and airway inflammation, remodeling, and hyperreactivity assessed. Mice were treated with anti-IL-33 or apyrase to neutralize or block IL-33 release. PVM infection or CRE exposure alone did not induce disease, whereas PVM/CRE coexposure acted synergistically to induce the hallmark features of asthma. CRE exposure during viral infection in early life induced a biphasic IL-33 response and impaired IFN-α and IFN-λ production, which in turn increased epithelial viral burden, airway smooth muscle growth, and type 2 inflammation. These features were ameliorated when CRE-induced IL-33 release was blocked or neutralized, whereas substitution of CRE with exogenous IL-33 recapitulated the phenotype observed in PVM/CRE-coexposed mice. Mechanistically, IL-33 downregulated viperin and interferon regulatory factor 7 gene expression and rapidly degraded IL-1 receptor-associated kinase 1 expression in plasmacytoid dendritic cells both invivo and invitro, leading to Toll-like receptor 7 hyporesponsiveness and impaired IFN-α production. We identified a hitherto unrecognized function of IL-33 as a potent suppressor of innate antiviral immunity and demonstrate that IL-33 contributes significantly to the synergistic interplay between respiratory virus and allergen exposures in the onset and progression of asthma.

P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate.

Prostate smooth muscle tone and hyperplastic growth are involved in the pathophysiology and treatment of male lower urinary tract symptoms (LUTS). Available drugs are characterized by limited efficacy. Patients’ adherence is particularly low to combination therapies of 5α-reductase inhibitors and α1-adrenoceptor antagonists, which are supposed to target contraction and growth simultaneously. Consequently, molecular etiology of benign prostatic hyperplasia (BPH) and new compounds interfering with smooth muscle contraction or growth in the prostate are of high interest. Here, we studied effects of p21-activated kinase (PAK) inhibitors (FRAX486, IPA3) in hyperplastic human prostate tissues, and in stromal cells (WPMY-1). In hyperplastic prostate tissues, PAK1, -2, -4, and -6 may be constitutively expressed in catecholaminergic neurons, while PAK1 was detected in smooth muscle and WPMY-1 cells. Neurogenic contractions of prostate strips by electric field stimulation were significantly inhibited by high concentrations of FRAX486 (30 μM) or IPA3 (300 μM), while noradrenaline- and phenylephrine-induced contractions were not affected. FRAX486 (30 μM) inhibited endothelin-1- and -2-induced contractions. In WPMY-1 cells, FRAX486 or IPA3 (24 h) induced concentration-dependent (1–10 μM) degeneration of actin filaments. This was paralleled by attenuation of proliferation rate, being observed from 1 to 10 μM FRAX486 or IPA3. Cytotoxicity of FRAX486 and IPA3 in WPMY-1 cells was time- and concentration-dependent. Stimulation of WPMY-1 cells with endothelin-1 or dihydrotestosterone, but not noradrenaline induced PAK phosphorylation, indicating PAK activation by endothelin-1. Thus, PAK inhibitors may inhibit neurogenic and endothelin-induced smooth muscle contractions in the hyperplastic human prostate, and growth of stromal cells. Targeting prostate smooth muscle contraction and stromal growth at once by a single compound is principally possible, at least under experimental conditions.

MP44-18 INHIBITION OF SMOOTH MUSCLE CONTRACTION BY THE INHIBITOR FOR CYTOHESIN FAMILIY GUANOSINE NUCLEOTIDE EXCHANGE FACTORS, SECIN H3 IN THE HYPERPLASTIC HUMAN PROSTATE

INTRODUCTION AND OBJECTIVES: In patients with benign prostatic hyperplasia, smooth muscle contraction and stromal growth in the prostate may both contribute to urethral obstruction and voiding symptoms. Although a context between control of smooth muscle tone and prostate growth may be assumed, molecular mechanisms underlying this connection still represent an enigma. Here, we addressed the role of Src family kinases (SFKs) for contraction of human prostate smooth muscle, and for growth of human stromal cells. METHODS: SFK expression was assessed by RT-PCR, Western blot, and immunofluorescence. Effects of two different SFK inhibitors, AZM475271 and PP2, on contraction were assessed in human prostate tissues obtained from radical prostatectomy. Effects on proliferation, actin organization, and cytotoxicity were examined in cultured stromal cells (WPMY-1). RESULTS: C-Src and Lck were detected by RT-PCR and Western blot in human prostate tissues and WPMY1 cells. Colocalization with calponin in immunofluorescence stainings of prostate sections indicated location to smooth muscle cells. AZM475271 (10 mM) and PP2 (10 mM) inhibited SFK activity in prostate tissues and WPMY-1 cell, being assessed as phospho-SFK content. Both inhibitors significantly inhibited contraction of prostate strips by norepinephrine, by the a1-adrenergic agonist phenylephrine, and by electric field stimulation. This may be explained by cytoskeletal deorganization, which was observed by phalloidin staining of actin filaments in response to AZM475271 and PP2 in WPMY-1 cells. This was paralleled by reduced proliferation of WPMY-1 cells, as indicated by EdU assay. Cytotoxicity was mainly observed at higher concentrations (50 mM or more) and longer incubation, but was minor at 10 mM for 24 h. CONCLUSIONS: Synchronized regulation of smooth muscle tone and growth by SFK-dependent control may explain their concomitant contributions to bladder outlet obstruction in the hyperplastic prostate. Targeting smooth muscle contraction and growth in the prostate at once by single agents may be principally possible.

Functionalized polyethylene naphthalate for cytocompatibility improvement

This work is focused on the characterization of functionalized polyethylene naphthalate (PEN) with Ar plasma treatment. The changes of the surface contact angle and the surface free energy, the surface morphology and roughness were studied using electrokinetic analysis, goniometry, AFM and XPS method. Ablation of PEN surface layers was determined by gravimetry. Plasma treated samples were consequently metallized; the surface morphology and cross-section of polymer/metal were studied with SEM and FIB-SEM analysis. The plasma treatment induced globular surface morphology connected with significant mass loss and wettability (contact angle) changes. Electrokinetic analysis confirmed dramatic changes in surface chemistry after plasma treatment depending on plasma power and exposure time. Also the ablated polymer mass increased with the increasing power and time. The plasma treatment increases oxygen concentration on the PEN surface. Consequently deposited gold layer and its FIB-SEM analysis confirmed homogeneous electrically continuous layer formation for a wide interval of plasma power and time used for the pretreatment. Plasma treatment was applied for significant enhancement of vascular smooth muscle growth cell adhesion and proliferation on PEN.