Muscle Cell Proliferation Research Articles

Abstract 508: Endothelial Cell Telomerase Promotes Aberrant Cell Proliferation in Pulmonary Hypertension

Background: Pulmonary arterial hypertension (PAH) is a progressive, severe disease with high morbidity and mortality. Mechanistically, early pulmonary vasoconstriction promotes cell proliferation and extensive vascular remodeling. Mammalian cell proliferation is dependent on telomerase expression; however, a mechanistic link between endothelial cell telomerase reverse transcriptase (TERT) and the control of mitogenic responses during the development of PAH has never been established. Hypothesis: Elevated telomerase levels in the diseased pulmonary endothelium induce proliferation of endothelial and smooth muscle cells in the pulmonary vasculature. Methods: We first received isolated pulmonary arterial endothelial cells from control subjects and patients with PAH. Autopsy sections obtained from patients were stained for endothelial cells, mitogenic markers, and TERT. Pooled primary pulmonary endothelial cells were used for in vitro experiments. Results: TERT expression was increased in the endothelium of pulmonary artery specimens of patients with PAH. Co-staining for the mitogenic marker PCNA confirmed high TERT expression specifically in proliferating cells. Key mechanistic stimuli causally involved in PAH, including hypoxia, inflammation, and vascular injury, increased mRNA and protein expression of TERT in pulmonary artery endothelial cells. TERT expression is necessary and sufficient for endothelial cell proliferation, as demonstrated through genetic TERT depletion and overexpression. Mechanistically, TERT supports the transcription of key endothelial growth factors, including vascular endothelial growth factor and epidermal growth factor, an effect that is mediated through TERT-dependent regulation of the transcription factor HIF1α. Conclusions: These data support the hypothesis that TERT promotes a proliferative phenotype of endothelial cells in PAH. Future experiments will investigate the cell-specific role of TERT in endothelial cell function and proliferation during the development of PAH using Cre/loxP recombination in mice harboring floxed TERT alleles.

Abstract 306: Endothelial 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase, Isoform 3 (PFKFB3) Deficiency Inhibits Hypoxia-Induced Pulmonary Hypertension in Mice

Background: Pulmonary artery hypertension (PAH) is a severe proliferative disease characterized by the remodeling of small pulmonary arteries leading to a progressive increase in pulmonary vascular resistance and ultimately to right ventricular failure and death. Many studies have indicated that endothelial dysfunction is a key element in the pathogenesis of this disease. In this study, we investigated whether endothelial glycolysis plays a critical role in the development of hypoxia-induced PAH by knocking down endothelial PFKFB3 (encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3), a critical regulator of glycolysis. Methods and Results: Immunostaining results showed that pulmonary artery endothelial PFKFB3 was increased in the lung of PAH patients. Mice deficient in endothelial Pfkfb3 showed decreased right ventricular systolic pressure, attenuated right ventricular hypertrophy and normal morphology of distal pulmonary arteries after four weeks of hypoxia compared with control mice. Furthermore, PFKFB3-deficient pulmonary arterial ECs secreted less growth factors, which ameliorated the proliferation of pulmonary artery smooth muscle cells. Additionally, the levels of endothelial derived inflammatory cytokines were also decreased. Besides, the inhibitory effect of endothelial Pfkfb3 supression on PAH formation were demonstrated in Sugen 5416/hypoxia rat pulmonary hypertension model with 3PO, a specific PFKFB3 inhibitor. Mechanistically, genetic deletion of PFKFB3 or 3PO treatment decreased the TNF-α induced NLRP3 inflammasome activation in pulmonary arterial ECs. Conclusions: Endothelial PFKFB3 inhibition is able to ameliorate the development of hypoxia-induced pulmonary hypertension.

Abstract 505: Targeting FOXM1 in Vascular Smooth Muscle Cells Induces Apoptotic Cell Death

Objectives: Vascular smooth muscle cell (VSMC) proliferation and dedifferentiation are key contributors to the initiation and progression of vascular diseases including restenosis. Forkhead Box M1 (FOXM1) is a proliferation-associated transcription factor shown to play a role in a variety of biological processes including cell cycle progression, cell survival, and apoptosis in many cell types. However, the role of FOXM1 in VSMC phenotypic transformation as a result of vascular injury following balloon angioplasty has not been studied. We hypothesize that FOXM1 modulates VSMC response by enhancing pro-proliferative and pro-survival signaling following vascular injury. Methods & Results: The rat carotid artery balloon injury was used to model vascular injury. Immunofluorescence staining was carried out on sections from injured carotid arteries or uninjured controls collected at 3, 7, or 14 days post injury (dpi). We observed that FOXM1 expression was upregulated in injured arteries at 7 and 14 dpi. Expression of FOXM1 in injured arteries co-localized with PCNA, a marker of proliferation. The upregulation of FOXM1 protein was replicated in cultured rat VSMCs by serum stimulation. Using thymidine synchronization, we observed that FOXM1 expression followed a cell cycle specific pattern in vitro . Chemical inhibition of FOXM1 using thiostrepton or FDI-6, or via siRNA resulted in decreased VSMC viability as measured by CCK-8, induction of apoptosis as measured by flow cytometry, and cleaved caspase 3 induction. More detailed immunocytochemistry analysis revealed that FDI-6 treated VSMCs exhibit disorganized microtubule networks. Furthermore, FDI-6 treatment decreased expression of Eg5, a motor protein essential for microtubule orientation and critical for mitosis execution. Additionally, FOXM1 inhibition decreased levels of β-catenin, a known activator of cell cycle-related genes and proliferation. Conclusions: In conclusion, our data suggest FOXM1 is critical to VSMC viability and may modulate VSMC proliferation and survival via regulation of important cell cycle and pro-proliferative proteins, and therefore may serve as a novel therapeutic target to prevent VSMC pathophysiology in the context of restenosis.

Abstract 507: Dedicator of Cytokinesis 2 Regulates Smooth Muscle Cell Proliferation via p38 Mitogen-Activated Protein Kinase Signaling Pathway

Smooth muscle cell (SMC) proliferation is an important process during vascular development and involved in vascular remodeling of a number of cardiovascular diseases including atherosclerosis, hypertension, and restenosis after angioplasty. However, the underlying mechanisms are not completely understood. In the present study, we identified dedicator of cytokinesis 2 (DOCK2) as one of the factors mediating the SMC proliferation. Knockout of DOCK2 (DOCK2-/-) caused abnormal blood vessel development in yolk sac and hemorrhage in mouse embryos. In addition, dorsal aorta wall structure was disrupted in DOCK2-/- embryos, and the SMC numbers in the dorsal aorta were decreased by 49% compared with wild type (WT). Consistently, proliferating cell nuclear antigen (PCNA)-positive cells in DOCK2-/- dorsal aorta media were much less the WT. These data suggest that the abnormal vascular development in DOCK2-/- embryos was due to the defective SMC proliferation. In vitro, platelet-derived growth factor (PDGF)-BB and serum upregulated DOCK2 along with PCNA expression in SMCs. Knockout or knockdown of DOCK2 by its shRNA inhibited the PDGF-BB-induced PCNA expression while forced expression of DOCK2 induced PCNA expression in SMCs. 5-ethynyl-2'-deoxyuridine (EdU) assay showed that knockdown of DOCK2 blocked PDGF-BB-induced SMC proliferation. Mechanistically, DOCK2 induced SMC proliferation by activating p38 mitogen-activated protein kinase (MAPK). Overexpression of DOCK2 induced while knockdown of DOCK2 inhibited p38 phosphorylation. Blockade of p38 MAPK signaling by its pathway-specific inhibitor diminished DOCK2-induced SMC proliferation. In a rat carotid artery balloon-injury model, knockdown of DOCK2 by adenoviral delivery of its shRNA blocked the injury-induced neointima formation and attenuated the PCNA expression. Furthermore, deletion of DOCK2 blocked ligation-induced neointima formation and PCNA expression in mouse arteries. Taken together, our studies demonstrated that DOCK2 is an important factor regulating SMC proliferation during embryo development as well as vascular remodeling in pathological conditions.

Abstract 677: Genetic Depletion of the Long Non-coding RNA H19 in Mice Protects from Elastase-induced Abdominal Aortic Aneurysms

Long noncoding RNAs (lncRNAs) have been shown as crucial molecular regulators in various biological processes and diseases. Recently we demonstrated that lncRNA H19 is highly upregulated during abdominal aortic aneurysm (AAA) development and progression in murine models (Angiotensin II in ApoE-/- mice; porcine pancreatic elastase model (PPE) in C57BL/6 mice). Experimental H19 knock-down using specific antisense LNA oligonucleotides showed a significant reduction in AAA growth in both models. Aim of this current study was to utilize genetically mutated H19-depleted mice (H19-/-) vs. wildtype littermate controls, to assess their behavior upon experimental AAA induction using PPE. In addition, we studied the proliferation rates of smooth muscle cells, originating from either H19-/- or H19+/+ mice in a kinetic live-cell imaging system. H19-/- on a C57BL/6J background were exposed to PPE. The aortic diameter in H19-/- mice was compared to WT littermate controls (upon PPE-AAA induction) at baseline, and then consecutively at days 7, 14, and 28. Primary mouse aortic smooth muscle cells were isolated from wild type or H19-depleted aortas, and cultured and monitored in the IncuCyte live cell imaging system for 48 hours, in an effort to study their proliferation rate. H19-/- mice upon PPE-AAA induction displayed significantly lower diameters throughout the study compared to WT controls. Primary aortic smooth muscle cells from H19-depleted mice showed greatly increased proliferation rates (based on cell confluency detection) in our kinetic live-cell imaging system in comparison to WT control cells. In conclusion, our study in H19-depleted mice supports our previously presented efforts, that H19 is an important contributor to experimental AAA development and progression. Further mechanistic studies will have to reveal the molecular properties of this long non-coding RNA in smooth muscle cell survival and proliferation.

Abstract 705: Nudt6 Inhibition Limits Smooth Muscle Cell Apoptosis and Abdominal Aortic Aneurysm Progression

Abdominal Aortic Aneurysm (AAA) is a highly lethal disease, of which diagnosis and treatment is still a major burden. Natural Antisense Transcripts (NAT) are known to inhibit key transcripts by antisense targeting. Their contribution to the development and progression of aneurysm disease has not been studied to date. The aim of this study was to investigate the role of the NAT Nudix Hydrolase 6 (Nudt6) and its antisense target Fibroblast growth factor (Fgf2) in AAA disease, by modulating NUDT6 in vivo in Angiotensin II (AngII)-induced experimental AAA via sonoporation (microbubble procedure) as well as in vitro in primary mouse aortic smooth muscle cells (mAoSMCs) via transfection . Further, primary mAoSMCs were observed using the IncuCyte imaging system to monitor changes in dynamic processes like proliferation and apoptosis. FGF2 is known to be a potent mitogen of smooth muscle cell (SMC) proliferation; a process being drastically diminished in AAA progression. By inhibiting Nudt6 with GapmeR in vivo we could observe smaller aortic diameters, increased both Fgf2 expression and SMCs survival by immunohistochemistry. In vitro , proliferation was increased in Nudt6 inhibited AngII-stimulated primary mouse aortic SMCs. Overexpression of Nudt6 did not have a significant effect on proliferation. Nudt6 and Fgf2 gene expression are inversely correlated and experience a switch of expression patterns during AngII stimulation over a longer period of time if primary SMCs are transfected with Nudt6 inhibitor and treated with AngII. All findings suggest a beneficial effect of Nudt6 inhibition in experimental AAA. In the future, it could be used as a potential therapeutic target in AAA by increasing SMC survival rates.

Effect of \u03b2-hydroxy-\u03b2-methylbutyrate on miRNA expression in differentiating equine satellite cells exposed to hydrogen peroxide

BackgroundSkeletal muscle injury activates satellite cells to initiate processes of proliferation, differentiation, and hypertrophy in order to regenerate muscle fibers. The number of microRNAs and their target genes are engaged in satellite cell activation. β-Hydroxy-β-methylbutyrate (HMB) is known to prevent exercise-induced muscle damage. The purpose of this study was to evaluate the effect of HMB on miRNA and relevant target gene expression in differentiating equine satellite cells exposed to H2O2. We hypothesized that HMB may regulate satellite cell activity, proliferation, and differentiation, hence attenuate the pathological processes induced during an in vitro model of H2O2-related injury by changing the expression of miRNAs.MethodsEquine satellite cells (ESC) were isolated from the samples of skeletal muscle collected from young horses. ESC were treated with HMB (24 h) and then exposed to H2O2 (1 h). For the microRNA and gene expression assessment microarrays, technique was used. Identified miRNAs and genes were validated using real-time qPCR. Cell viability, oxidative stress, and cell damage were measured using colorimetric method and flow cytometry.ResultsAnalysis of miRNA and gene profile in differentiating ESC pre-incubated with HMB and then exposed to H2O2 revealed difference in the expression of 27 miRNAs and 4740 genes, of which 344 were potential target genes for identified miRNAs. Special attention was focused on differentially expressed miRNAs and their target genes involved in processes related to skeletal muscle injury. Western blot analysis showed protein protection in HMB-pre-treated group compared to control. The viability test confirmed that HMB enhanced cell survival after the hydrogen peroxide exposition.ConclusionsOur results suggest that ESC pre-incubated with HMB and exposed to H2O2 could affect expression on miRNA levels responsible for skeletal muscle development, cell proliferation and differentiation, and activation of tissue repair after injury. Enrichment analyses for targeted genes revealed that a large group of genes was associated with the regulation of signaling pathways crucial for muscle tissue development, protein metabolism, muscle injury, and regeneration, as well as with oxidative stress response.

Effect of emodin on the proliferation and migration of hypoxia pulmonary artery smooth muscle cells

Objective To observe the inhibitory effect of emodin on the proliferation and migration of pulmonary artery smooth muscle cells. Methods Primary cultured human pulmonary arterial smooth muscle cells (HPASMC) were cultured in a routine manner to establish an hypoxic cell model. Emodin at different concentrations (0, 5, 10, 15 and 20 μmol/L) 48 and 72 h later, the proliferation and migration of hypoxic pulmonary artery were detected by methyl thiazol tetrazolium (MTT) assay and Transwell invasion assay. Western blotting assay was used to determine the expression of p38 protein in hypoxia HPASMC induced by emodin. Results Emodin inhibited the proliferation and migration of hypoxic HPASMC in a time-and concentration-dependent manner. When the concentration was 20 μmol/L, the effect was the most significant, and the inhibitory rates at 12, 24, 48 and 72 h were (53.46±1.81)%, (61.32±2.69)%, (76.34±3.05)% and (84.43±3.18)%, respectively. (68.90±8.49), (46.10±5.63), (27.56±5.38) in the Transwell experimental group (5, 10, 15 and 20 μmol/[(12.61±3.68)] compared with the control group [(88.59±8.56)]. Compared with the control group, the experimental group p38 protein expression was reduced (P=0.001). Conclusion Emodin can significantly inhibit the proliferation and migration of human pulmonary artery smooth muscle cells under hypoxic conditions and down-regulate the expression of p38 protein. Key words: Emodin; Pulmonary artery smooth muscle cells; Proliferation; Migration

Identification and Characterization of Protein Phosphatase Methylesterase 1 (Ppme1) in Skeletal Muscle

Skeletal muscle atrophy results from a variety of physiological conditions, including immobilization, denervation, glucocorticoid treatment, cancer, and aging. In order to elucidate a better understanding of the molecular and genetic mechanisms of skeletal muscle atrophy, a microarray study was performed using muscle tissue isolated from mice undergoing neurogenic atrophy in response to denervation. The data generated from this microarray identified a suite of several hundred predicted genes that displayed differential expression under conditions of muscle wasting, including protein phosphatase methylesterase 1 (Ppme1). The Ppme1 gene was observed to be transcriptionally upregulated in response to neurogenic skeletal muscle atrophy (i.e. denervation). To confirm that Ppme1 is expressed in muscle, we successfully cloned the predicted Ppme1 cDNA from mouse muscle cells. Additionally, Ppme1 mRNA expression levels were analyzed by quantitative polymerase chain reaction (qPCR) and found to be highly expressed in both proliferating and differentiated muscle cells. Furthermore, Ppme1 protein levels were analyzed by Western blot using protein homogenates isolated from muscle cells and Ppme1 was found to be ubiquitously and uniformly expressed at all time points throughout muscle cell proliferation and differentiation. In order to assess the transcriptional activity of the Ppme1 gene, a fragment of the promoter was cloned, fused to a reporter gene, transfected into C2C12 cells, and found to be transcriptionally active. Additionally, the Ppme1 reporter gene was transfected in combination with MyoD1 and myogenin expression plasmids and differential effects on the Ppme1 reporter gene activity were observed in response to myogenic regulatory factor ectopic expression. The results presented here help contribute new insight into the molecular and genetic mechanisms of muscle atrophy and help provide a better understanding of the genome‐wide transcriptional changes that occur during neurogenic atrophy‐induced muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants to D.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Essential role of smooth muscle Rac1 in airway hyperresponsiveness and airway remodelling associated to severe asthma

Introduction Severe asthma is a lung chronic disease characterized by inflammation, airway hyperresponsiveness (AHR), airway remodelling and corticoid resistance. The molecular mechanisms underlying airway smooth muscle cells (aSMC) contraction and proliferation involved in airway hyperresponsiveness (AHR) and airway remodelling are still largely unknown. Small GTPases of the Rho family (RhoA, Rac1 and Cdc42) play a central role in smooth muscle functions. Recently, we demonstrated that Rac1 play a major role in the control of bronchoconstriction by modulating airway SMC contraction (Andre-Gregoire et al., J Allergy Clin Immunol, 2017). The objective of this study was to assess the role of smooth muscle Rac1 in severe asthma. Methods We developed a new model of severe allergic asthma (house dust mite (HDM) sensitized mice) characterized by AHR and strong airway remodelling, including SMC hyperplasia. Interestingly, as in human severe asthma, this model is associated to an altered sensitivity to inhaled corticoids. To assess the role of Rac1 in aSMCs, SMC-Rac1-KO mice were sensitized to HDM. Results Our results demonstrated that deletion of SMC Rac1 totally prevents AHR and smooth muscle mass increase in this murine model. Furthermore, nebulisation of the Rac1 inhibitor NSC23766 prevents AHR and SMC hyperplasia, but also goblet cell hyperplasia and epithelial cell hypertrophy whereas corticoids treatments has no effect on these parameters. In addition, we observed that nebulisation of NSC23766 decreased eosinophil and neutrophil populations in bronchoalveolar lavages from HDM sensitized asthmatic mice. Discussion In this study, we generated a new model of severe allergic asthma that mimic closely the human pathology. We demonstrated that Rac1 plays an essential role in aSMC contraction and hyperplasia, and that its inhibition prevents AHR and airway remodelling associated to severe asthma. Compared to classical bronchodilators, Rac1 inhibition presents the additional advantage to decrease pulmonary inflammation. Conclusion Consistant with these datas, inhibition of Rac1 activity may represent a novel therapeutic approach for patients with severe asthma.

Inhibitory effects of solid lipid nanoparticles of rhynchophylline on proliferation of rat vasculars mooth muscle cells induced by TGF-β1

Objective To observe the effect of Rhy-SLN on the proliferation of rat vascular smooth muscle cells (VSMC) induced by TGF-β1, and explore the mechanism. Methods The primary culture of rat thoracic aortic vascular smooth muscle cells was studied by tissue block culture method. The cells were divided into the control group, TGF-β1 group, TGF-β1+ the high, medium and low dosage groups of Rhy-SLN. In addition to the control group, the cells of the other groups were involved in the intervention of TGF-β1 of 20 g/L, and the high, medium and low dosage groups of Rhy-SLN cells were involved in the intervention of 25, 50, 100 mg/L of the hook teng solid lipid nanoparticles. After 24 hours of culture, MTT assay was used to detect cell proliferation inhibition rate in each group, and the cell cycle was detected by flow cytometry. The expression of c-myc and c-Fos protein in each group was detected by Western blot method. Results Compared with the TGF-β1 group, the absorbance value (0.457 ± 0.046 vs. 0.975 ± 0.049) of TGF-β1+ rhy-sln high dose group significantly decreased (P<0.01); the number of S phase cells (15.87% ± 2.47%, 15.23% ± 1.69%, 17.02% ± 2.87% vs. 38.58% ± 2.68%) of TGF-β1+ rhy-sln in each dose group significantly decreased (P<0.01); The c-myc (48.65 ± 3.65, 50.69 ± 4.16, 55.29 ± 3.67 vs. 68.21 ± 3.25) and c-Fos (38.78 ± 4.25, 43.56 ± 3.69, 46.58 ± 3.57 vs. 66.54 ± 4.09) of the TGF-β1+ rhy-sln each dose group significantly decreased (P<0.01). Conclusions The Rhy-SLN can inhibit the proliferation of VSMC in rats induced by TGF-β1.Its mechanism is related to the conversion of G0/G1 phase to the S phase and the expression of the reduction of c-myc and c-fos protein. Key words: Rhynchophylline; Solid lipid nanoparticles; Transforming growth factor beta; Proto-oncogene proteins c-fos; Proto-oncogene proteins c-myc; Muscle, smooth, vascular; Cell proliferation; Rat

Eosinophil-derived exosomes contribute to asthma remodelling by activating structural lung cells.

Eosinophils, a central factor in asthma pathogenesis, have the ability to secrete exosomes. However, the precise role played by exosomes in the biological processes leading up to asthma has not been fully defined. We hypothesized that exosomes released by eosinophils contribute to asthma pathogenesis by activating structural lung cells. Eosinophils from asthmatic patients and healthy volunteers were purified from peripheral blood, and exosomes were isolated from eosinophils of asthmatic and healthy individuals. All experiments were performed with eosinophil-derived exosomes from healthy and asthmatic subjects. Epithelial damage was evaluated using primary small airway epithelial cell lines through 2 types of apoptosis assays, that is, flow cytometry and TUNEL assay with confocal microscopy. Additionally, the epithelial repair was analysed by performing wound healing assays with epithelial cells. Functional studies such as proliferation and inhibition-proliferation assays were carried out in primary bronchial smooth muscle cell lines. Also, gene expression analysis of pro-inflammatory molecules was evaluated by real-time PCR on epithelial and muscle cells. Lastly, protein expression of epithelial and muscle cell signalling factors was estimated by Western blot. Asthmatic eosinophil-derived exosomes induced an increase in epithelial cell apoptosis at 24 hour and 48 hour, impeding wound closure. In addition, muscle cell proliferation was increased at 72 hours after exosome addition and was linked with higher phosphorylation of ERK1/2. We also found higher expression of several genes when both cell types were cultured in the presence of exosomes from asthmatics: CCR3 and VEGFA in muscle cells, and CCL26, TNF and POSTN in epithelial cells. Healthy eosinophil-derived exosomes did not exert any effect over these cell types. Eosinophil-derived exosomes from asthmatic patients participate actively in the development of the pathological features of asthma via structural lung cells.

Cyclic stretch-induced Crp3 sensitizes vascular smooth muscle cells to apoptosis during vein arterialization remodeling.

Vein graft failure limits the long-term patency of the saphenous vein used as a conduit for coronary artery bypass graft. Early graft adaptation involves some degree of intima hyperplasia to sustain the hemodynamic stress, but the progress to occlusion in some veins remains unclear. We have demonstrated that stretch-induced up-regulation of cysteine and glycine-rich protein 3 (Crp3) in rat jugular vein and human saphenous vein in response to arterialization. Here, we developed a Crp3-KO rat to investigate the role of Crp3 in vascular remodeling. After 28 days jugular vein arterialization, the intima layer was 3-fold thicker in the Crp3-KO that showed comparable smooth muscle cells (SMC) proliferation but an absence of early apoptosis observed in the wild-type rat (WT). We then investigated the role of Crp3 in early integrin-mediated signaling apoptosis in isolated jugular SMC. Interestingly, under basal conditions, ceramide treatment failed to induce apoptosis in both WT and Crp3-KO SMC. Under stretch, Crp3 expression increased in WT SMC and ceramide induced apoptosis. Immunoblotting analysis indicated that ceramide stretch-induced apoptosis in SMC is accompanied by a decrease in the phosphorylation status of both Fak and Akt, leading to an increase in Bax expression and caspase-3 cleavage. In contrast, ceramide failed to decrease Fak and Akt phosphorylation in Crp3-KO SMC and, therefore, there was no downstream induction of Bax expression and effector caspase-3 cleavage. Taken together, we provide evidence that stretch-induced Crp3 modulates vein remodeling in response to arterialization by sensitizing SMC to apoptosis.

Radiation suppresses neointimal hyperplasia through affecting proliferation and apoptosis of vascular smooth muscle cells.

To study the effect of x-ray radiotherapy on vascular smooth muscle cells (VSMCs) and elucidate the mechanisms in preventing neointimal hyperplasia of prosthetic vascular grafts. In model I, twelve mongrel dogs underwent revascularization with prosthetic grafts and half the dogs underwent irradiation of the grafts at 28 Gy. In model II, human VSMCs (hVSMCs) were maintained and divided into six groups to which external radiation was applied at six different doses: 0 Gy, 2 Gy, 8 Gy, 16 Gy, 24 Gy and 30 Gy. In both models, specimens were harvested and examined by using morphological, immunological, cellular and molecular methods. After irradiation, the neointima thickness was significantly lower in irradiated groups (p≤0.01). The radiotherapy could up-regulate p27kip1, and down-regulate proliferating cell nuclear antigen (PCNA) and S phase kinase associated protein 2 (Skp2). X-ray irradiation inhibits the proliferation of hVSMCs via acting on G1/S phase of cell cycle. The apoptosis of hVSMCs increased significantly with dose and time. The expression of PCNA and Skp2 were decreased after a first increasing trend with dose, but had a significant negative correlation with time. The expression of p27kip1 had a significant positive correlation with dose and time. Postoperative external fractionated irradiation after prosthetic vessel replacement of the abdominal aorta suppressed the development of hyperplasia in the graft neointima in the short term. There was a prominent time- and dose-dependent inhibition of VSMC proliferation by radiation when it was administered.

The Different Effect of R and S Albuterol on Proliferation and Migration of Airway Smooth Muscle Cells

studies have shown that the R- and S-enantiomers of racemic albuterol, a β2-adrenergic receptor agonist used in asthma treatment, have differential effects on the contractile properties of airway smooth muscle. However, the effect of albuterol on the proliferation and migration has never been tested. Since (R)-, but not (S)-albuterol enhances bronchodilation, we expect the two racemic isomers would also affect proliferation and migration of tracheal cells differentially. By monitoring migratory properties of airway smooth muscle cells in the presence of albuterol isomers, the different effect of albuterol on proliferation and migration of airway smooth muscle cells is probed. The results show Both of R- and S-albuterol could inhibit the proliferation of smooth muscle cells and the inhibition ratio of these two isomers had no significant difference; R-albuterol, but not S-albuterol, inhibited cell migration.

Screening of ordinary commercial varieties of apple fruits under different storage conditions for their potential vascular and metabolic protective properties.

Epidemiological studies reported that apple consumption is associated with a decrease of cardiovascular and metabolic dysfunction, probably due to the polyphenols and fibers present in this fruit. The storage conditions and genetic origin of apples have been reported to influence their content and, as a consequence, their pharmacological properties. The present study evaluated the influence of varieties and storage conditions of traditional and highly appreciated apples including Gala, Golden Delicious, Granny Smith and Pink Lady varieties after harvest and storage under classic cold conditions, under a controlled atmosphere, or under extreme ultra-low oxygen conditions. Thus, a multi-parametric screening on cell models associated with vascular and metabolic dysfunctions - such as endothelial and smooth muscle cells, hepatocytes, adipocytes and macrophages - in relation to the apple polyphenol content has been developed. This strategy demonstrated that, overall, peeled apple samples exhibited a vascular tropism and acted mainly on proliferation and oxidative stress in endothelial and smooth muscle cells. Apple extracts appeared to be less effective on adipocytes and macrophages, but they exhibited antioxidant properties in hepatocytes. Among the varieties, Gala and Golden Delicious were the most efficient against the processes involved in the development of atherosclerosis. Concerning storage conditions, most of the apple varieties were more efficient under harvest conditions, while they could not be discriminated under all other cold conditions and the concentration used, except for the Gala samples. Interestingly, pharmacological properties were associated with the polyphenol profiles of freeze dried apple flesh powder. The present report revealed the potential use of some apple extracts as effective food supplements or nutraceuticals for the prevention and/or management of cardiovascular and metabolic diseases.

Nitric oxide and heart disease: New discoveries and innovations in diagnostics and therapeutics

N oxide (NO) is a multifunctional signaling molecule, intricately involved with maintaining a host of physiological processes including but not limited to host defense, neuronal communication and the regulation of vascular tone. The endothelium-derived NO plays a crucial role in regulating a wide spectrum of functionsin the cardiovascular system, including vasorelaxation, inhibition of leukocyte-endothelial adhesion, vascular smooth muscle cell (SMC) migration and proliferation, as well as platelet aggregation. In this regard, NO is a potent vasodilator as well as a powerful anti-platelet and anti-leukocyte factor. NO is one of the most important signaling molecules in our body. Loss of NO function is one of the earliest indicators or markers of disease. Experimental and clinical studies provide evidence that defects of endothelial NO production, referred to as endothelial dysfunction, is not only associated with all major cardiovascular risk factors such as hyperlipidemia, diabetes, hypertension, erectile dysfunction, smoking and severity of atherosclerosis, but also has a profound predictive value for future atherosclerotic disease progression. Emerging published literature reveals that NO insufficiency may manifest itself differently in different patients. Thirty plus years after its discovery and over 14 years since a Nobel Prize was awarded for its discovery, there have been no hallmark therapeutic breakthroughs. We will review the current state of the science surrounding nitric oxide in the etiology of a number of different disease states and reveal the latest technology to safely and effectively restore nitric oxide in patients. The audience will learn the challenges and opportunities that exist in understand NO homeostasis in their patients and how this may translate into better management of their patients.

Caveolin-3 promotes glycometabolism, growth and proliferation in muscle cells.

ObjectiveCaveolin-3 (CAV3) protein is known to be expressed specifically in various myocytes, but its physiological function remains unclear. CAV3, located at the cell membrane, may promote the sensitivity of the Akt signaling pathway, which is closely related to glucose metabolism and to cell growth and proliferation.MethodsThe CAV3 gene was stably transfected into C2C12 muscle cells, and the effects were evaluated by biochemical assays, WB and confocal microscopy for the observation of cellular glucose metabolism, growth and proliferation, and the effect of CAV3 on the Akt signaling pathway with no insulin stimulation.ResultsAfter C2C12 cells were transfected with the mouse CAV3 gene, which increased CAV3 expression, the abundance of the CAV3 and GLUT4 proteins on the cell membrane increased, but the total GLUT4 protein content of the cell was unchanged. Glucose uptake was increased, and this did not affect the glycogen synthesis, but the cell surface area and cell proliferation increased. While there were significant increases in p-Akt and p-p70s6K, which is a downstream component of Akt signaling, the level of GSK3β protein, another component of Akt signaling did not change.ConclusionsThe muscle, CAV3 protein can activate Akt signaling, increase GLUT4 protein localization in the cell membrane, increase glucose uptake, and promote myocyte growth and proliferation. CAV3 protein has a physiological role in glycometabolism, growth and proliferation, independent of insulin stimulation.

Abstract 21217: PTP1B is a Critical Mediator for Vascular Smooth Cell Proliferation in the Development of Pulmonary Hypertension in vivo and in vitro

Background: Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling that leads to right ventricular failure and death. In PH, vascular remodeling is driven by hyper-proliferative and anti-apoptotic pulmonary arterial smooth muscle cells (PASMC). Protein Tyrosine Phosphatase 1B (PTP1B), is a negative regulator for apoptosis and proliferation of vascular smooth muscle cells through down-stream mediators such as BCL-2. While BCL-2 is increased in PH patients, the pathways for regulating BCL-2 are poorly understood. Therefore, our aim is to investigate if PTP1B has a role in PASMC proliferation and resistance to apoptosis in human PASMCs and in the angioproliferative Sugen/Hypoxia/Normoxia (SuHx) PH rat model. Method: Male Sprague-Dawley rats were treated with a single intraperitoneal dose of a vascular endothelial growth factor antagonist (SU5416, 20 mg/kg, n=4) and kept in hypoxia for 3 weeks followed by normoxia for 2 weeks. Saline treated controls were kept in normoxia for 5 weeks (n=4). RV systolic pressure (RVSP) was recorded by RV catheterization and hearts were isolated to assess RV hypertrophy (RV/LV + IVS). PTP1B and BCL-2 expression was determined using qPCR. Healthy hPASMCs were incubated with a PTP1B inhibitor (Ethyl-3,4-dephostatin) at IC50=0.58ug/ml for 24hrs and stained with Ki67 to assess for proliferation. Results: SuHx rats had severe PH, evident by elevated RVSP compared to control (88.97+/- 13.67 vs. 28.47+/- 2.22 mmHg, p&lt;0.05). PH rats also showed reduced RV function and increased hypertrophy (0.7+/- 0.063 vs 0.274 +/-0.01 in control, p&lt;0.05). PH lungs exhibited pulmonary vascular remodeling characterized by excessive growth of the PASMCs. PTP1B was decreased in PH lungs compared to controls (0.158+/-0.0647 vs 1+/-0.06, P&lt;0.05). As expected, BCL-2 expression was significantly increased in PH compared to control (2.01+/-0.162 vs 1 +/-0.1, P&lt;0.01). Inhibition of PTP1B in cultured hPASMCs increased proliferation by ~2 fold as assessed by Ki67 positive cells. Conclusion: Severe Angioproliferative PH in rats is associated with a downregulation of PTP1B and increased expression of BCL-2. Additionally, PTP1B inhibition resulted in hPASMC proliferation, suggesting a role for PTP1B in the pathogenesis of PH.

Abstract 24034: MFG-E8 Fragment Medin and Arterial Aging

Background: Aging increases milk fat globule EGF-VIII (MFG-E8) expression in the rat, nonhuman primate and human aortic walls, facilitating the invasion, proliferation, and proinflammation of vascular smooth muscle cells (VSMCs). The MFG-E8 C-terminal fragment medin has been reported to be involved in the necrosis of VSMCs at the inflammatory sites of aortic dissection, however, its cleavage processes, aortic wall levels with aging and bio-role in VSMCs remains to be determined. Material and Methods: In this study, immunofluorescence, immunohistochemistry and western blot analysis demonstrate that MFG-E8 and its fragment medin ( Figure ), activated matrix metalloproteinase type II (MMP-2) and platelet derived growth factor receptor type-beta (PDGFR-β) protein levels were markedly increased in human grossly normal aortic walls from old (n=10, &gt;50 yrs) vs. younger donors (n=10, &lt;25 yrs). Importantly, exposure of medin peptide (20 to100nM) to primary cultured VSMCs isolated from young (8 mo) and old (30 mo) FXBN rat aortae significantly increased MMP-2 activation, PDGFR-β expression, and migratory capacity measured by a modified Boyden chamber in a dose-dependent manner in both also. Separate exposure of activated MMP-2 to recombinant human MFG-E8 protein and also to MFG-E8 enriched old human aortic protein markedly increased the cleavage product medin in both, which was substantially inhibited by an MMP inhibitor, GM6001. Exposure of medin to VSMCs did not significantly affect the expression of cell cycle related proteins. In addition, PDGF-BB treatment markedly activated MMP-2 in both young and old VSMCs, which was substantially reduced by a PDGFR-β inhibitor, RTK. Conclusion: Taken together, targeting MFG-E8 or its cleavage product medin is a novel approach to the prevention or treatment of large arterial aging or age-associated disease.