GFP Adenovirus Research Articles

Abstract P2023: Mitogen-activated Protein Kinase-directed Modulation Of The Non-sarcomeric Actin Cytoskeleton In Cardiomyocytes

A better understating of the mechanisms by which cardiomyocytes grow in different directions could open up new therapeutic avenues for hypertrophic and dilated cardiomyopathies, diseases that include the widening and lengthening of cardiomyocytes, respectively. Mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinases 1/2 (ERK1/2) are known to control this directional growth of cardiomyocytes, such that activated MEK1 causes concentric hypertrophy and cardiomyocyte widening, while genetic ablation of ERK1/2 proteins causes dilation and cardiomyocyte lengthening. To determine how these proteins may control this directional growth response, we conducted a phosphoproteomic screen using neonatal rat ventricular myocytes (NRVMs) treated with activated MEK1 adenovirus, U0126 MEK1 inhibitor, or control GFP adenovirus. The resulting data pinpointed over 2700 sites on 324 proteins as changing their phosphorylation status with modulation of this signaling cascade. Gene ontology analysis found that cytoskeletal-associated proteins were the largest subset of proteins with phosphorylation changes. Traditional and PhosTag western blots verified that many of these cytoskeletal proteins change expression as well as their phosphorylation status in NRVMs. In vivo experiments showed that cytoplasmic actin expression was upregulated in MEK1 transgenic or ERK1/2 gene-deleted hearts. Furthermore, MEK1 transgenic adult cardiomyocytes had enhanced cytoplasmic actin at lateral membranes. Palladin, an actin nucleating protein, changed both phosphorylation status and isoform expression in response to modulation of MEK1-ERK1/2 signaling. Adeno-associated viruses were used to overexpress different palladin isoforms. Immunostaining for these proteins showed that localization of palladin isoforms moved from the subcortical compartment to the sarcomere, suggesting these isoforms preferentially associate with either cytoskeletal or sarcomeric actin. Future studies will further examine the signaling roles that cytoplasmic actin may play in healthy and diseased cardiomyocytes and delineate preferential binding of actin associated proteins between cytoplasmic and sarcomeric actin in cardiomyocytes.

Thymosin β4 regulates endothelial cell function via activating the AKT pathway

The vascular eendothelial cells are highly heterogeneous and associated with numerous diseases. Thymosin β4 (Tβ4) plays pleiotropic roles in endothelial cell differentiation, migration and angiogenesis. However, the underlying mechanisms played by Tβ4 in the regulation of endothelial cells have not yet been well investigated. In the present study, Tβ4 -GFP adenovirus, transfected into human umbilical vein endothelial cells (HUVECs), and cell morphology were analyzed by fluorescence microscopy. ELISA was used to determine the concentration of Tβ4 expression. Furthermore, the effects of Tβ4 overexpression on HUVECs proliferation, apoptosis and migration were investigated. Real-time quantitative PCR and western blot were conducted to examine mRNA and protein expression in HUVECs with Tβ4 overexpression. Moreover, the underlying molecular mechanism of Tβ4 in HUVECs function was tested through treatment with LY294002, a PI3K/AKT inhibitor. Overexpression of Tβ4 increased the cell ability of HUVECs, and up-regulated the expression of the proliferation markers PCNA and Cyclin D1. In addition, overexpression of Tβ4 reduced HUVECs apoptosis, both under normoxic and hypoxic conditions. Moreover, overexpression of Tβ4 increased the ability of HUVECs to migrate through the membrane and up-regulated levels of MMP-2 and MMP-9. The use of LY294002 decreased the p-AKT (Ser473) level, which was induced by Tβ4 overexpression. Importantly, LY294002 reduced Tβ4-induced HUVECs proliferation and migration. In conclusion, our results suggest that Tβ4 is a major regulator of HUVECs function by activating the AKT signaling pathway.

ECM1: A CTGF Binding Protein Inhibiting Ductular Reaction and Liver Fibrosis

IntroductionExtracellular matrix protein 1 (ECM1) has inhibitory roles against transforming growth factor (TGF)β activation and liver fibrosis. We have identified ECM1 as binding protein for connective tissue growth factor (CTGF) in yeast two‐hybrid analysis. This study aims to determine whether ECM1 binding inhibits CTGF‐mediated ductular reaction and liver fibrosis.MethodsMice were fed with 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) to induce ductular reaction and liver fibrosis. ECM1 was overexpressed in adenovirus and intravenously injected into animals before DDC feeding. ECM1 interaction with CTGF, and their effects on liver injury after DDC feeding were analyzed.ResultsECM1 downregulation was correlated with CTGF upregulation during DDC‐induced liver injury. ECM1 overexpression after DDC feeding caused low levels of EpCAM and Sox9 mRNAs, small areas of EpCAM+ cells, and few proliferating ductular epithelial cells in comparison to controls that received GFP adenovirus. Weak fibrogenesis was also observed in the Ecm1 overexpressing livers, as evidenced by low levels of fibrosis‐related genes, small fibrotic areas in staining with α smooth muscle actin antibody and Sirius red as well as low collagen production determined by hepatic hydroxyproline content. Finally, we found that ECM1 could interact with CTGF and integrin αvβ6 leading to low TGFβ activation in vitro and in vivo.ConclusionThese observations indicate that ECM1 is a novel binding protein for CTGF. Its overexpression significantly reduces DDC‐induced ductular reaction and fibrogenic reaction, supporting anti‐fibrotic potentials of this molecule for the treatment of chronic liver disease.Support or Funding InformationThis study is supported by National Institutes of Health NIAAA KO1AA024174 grant and Children Miracle Research Foundation grant awarded to L Pi.

2191-P: PPM1K Regulates ß-Cell Proliferation, Insulin Content, and GSIS

A single nucleotide polymorphism in the gene encoding the protein phosphatase m1K (PPm1K) is associated with increased risk for the development of type 2 diabetes but not insulin resistance in humans. We recently discovered that expression of the PPM1K gene in liver is suppressed by the carbohydrate response element binding protein (ChREBP) which controls transcriptional responses to changes in carbohydrate availability. Taken together these findings suggest that PPM1K might play an important role in the glucose responsive pancreatic β cell; however, little is known about the role of PPm1K in this cell type. Here we studied how changes in glucose concentration affect PPm1K expression in β cells and explored the effect of modulating PPM1K expression on β cell proliferation, insulin content and glucose stimulated insulin secretion. Studies were carried out in INS1-832/13 β cells in culture and key findings were validated in isolated rat islets. In line with our previous observations in liver, we found that increasing glucose concentrations in INS1 cells results in elevated ChREBP activity, measured by expression of the ChREBP β isoform, and lower PPM1K mRNA expression (P<0.01). Treatment of INS-1 cells with adenovirus expressing recombinant human PPM1K for 96h increased β cell proliferation (P<0.05) but robustly lowered GSIS (P<0.001). This effect was due to substantially lower insulin content in the Ad-PPm1K treated cells (P<0.01). Importantly, there was no effect of the control GFP adenovirus compared to non-transfected cells for any of these measures. Moreover, siRNA mediated knock down of PPM1K resulted in increased expression of INS1 and INS2 mRNA suggesting that altered transcription of the insulin gene likely accounts for the effect of PPm1K on β cell insulin content and release. In conclusion, our data clearly demonstrate that PPM1K plays an important role in the regulation of β cell insulin production and proliferation. Disclosure Y. Deleye: None. J. Herring: None. K.H. Hess: None. B.R. Leininger: None. S.A. Hannou: None. I. Astapova: None. M.A. Herman: None. J.S. Tessem: None. P.J. White: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-16-INI-17 to P.J.W.)

Eliminating contraction during culture maintains global and local Ca2+ dynamics in cultured rabbit pacemaker cells

Pacemaker cells residing in the sinoatrial node generate the regular heartbeat. Ca2+ signaling controls the heartbeat rate—directly, through the effect on membrane molecules (NCX exchange, K+ channel), and indirectly, through activation of calmodulin-AC-cAMP-PKA signaling. Thus, the physiological role of signaling in pacemaker cells can only be assessed if the Ca2+ dynamics are in the physiological range. Cultured cells that can be genetically manipulated and/or virally infected with probes are required for this purpose. Because rabbit pacemaker cells in culture experience a decrease in their spontaneous action potential (AP) firing rate below the physiological range, Ca2+ dynamics are expected to be affected. However, Ca2+ dynamics in cultured pacemaker cells have not been reported before. We aim to a develop a modified culture method that sustains the global and local Ca2+ kinetics along with the AP firing rate of rabbit pacemaker cells.We used experimental and computational tools to test the viability of rabbit pacemaker cells in culture under various conditions. We tested the effect of culture dish coating, pH, phosphorylation, and energy balance on cultured rabbit pacemaker cells function. The cells were maintained in culture for 48 h in two types of culture media: one without the addition of a contraction uncoupler and one enriched with either 10 mM BDM (2,3-Butanedione 2-monoxime) or 25 μM blebbistatin. The uncoupler was washed out from the medium prior to the experiments. Cells were successfully infected with a GFP adenovirus cultured with either BDM or blebbistatin. Using either uncoupler during culture led to the cell surface area being maintained at the same level as fresh cells. Moreover, the phospholamban and ryanodine receptor densities and their phosphorylation level remained intact in culture when either blebbistatin or BDM were present. Spontaneous AP firing rate, spontaneous Ca2+ kinetics, and spontaneous local Ca2+ release parameters were similar in the cultured cells with blebbistatin as in fresh cells. However, BDM affects these parameters. Using experimental and a computational model, we showed that by eliminating contraction, phosphorylation activity is preserved and energy is reduced. However, the side-effects of BDM render it less effective than blebbistatin.

Abstract 306: Identifying Downstream Effectors of the Extracellular Signal-related Kinase 1/2-controlled Directional Growth Response in Cardiomyocytes

A better understating of the mechanisms by which cardiomyocytes grow in different directions could open up new therapeutic avenues for hypertrophic and dilated cardiomyopathies, diseases that include the lengthening and widening of cardiomyocytes, respectively. To-date the only known proteins found to control this directional growth of cardiomyocytes are MEK1, mitogen-activated protein kinase kinase 1, and ERK1/2, extracellular signal-related kinase 1/2. The only known role of MEK1 is to phosphorylate ERK1/2 after MEK1 itself becomes phosphorylated through RAS/RAF signaling. Upon phospho-activation, ERK1/2 can interact with targets in the cytoplasm or translocate to the nucleus to induce cell growth and proliferation pathways. We previously found that cardiac overexpression of activated MEK1 causes cardiac hypertrophy and cardiomyocyte widening, while genetic ablation of ERK1/2 proteins in the heart causes dilation and cardiomyocyte lengthening. To determine how these proteins may control this directional growth response, we conducted a phosphoproteomic screen using neonatal rat ventricular myocytes (NRVMs) treated with activated MEK1 adenovirus, U0126 (a MEK1 inhibitor), or GFP adenovirus (control). For proteins with the greatest change in phosphorylation upon MEK1 activation or inhibition, we have verified the screen results using Phos-tag gels and western blots. Many of these hits have cytoskeletal or actin regulatory functions, and a few have also been implicated in stress-sensing in previous literature in other cells types. Furthermore, many hits showed increased protein levels in addition to augmented phosphorylation in NRVMs treated with activated MEK1 adenovirus. Given our nearly 20 interesting candidate proteins, we are currently conducting an in vivo screen in which each gene is individually expressed in mice using AAV9. After injection of virus into the thoracic cavities of P5 wild type mice, we are assessing if cardiac hypertrophy/dilation or cardiomyocyte dimensional growth are present at 4 months of age. Any novel candidates identified in this manner will be presented.

P-187 - Nox 5 overexpression induces the activation of the unfolded protein response in human aortic endothelial cells

Oxidative stress is a critical mechanism that underlies the pathophysiology of cardiovascular diseases. At the vascular level, an enhanced NADPH oxidase-mediated superoxide production associates with endothelial dysfunction, metalloprotease secretion, cellular senescence, and more recently activation of the unfolded protein response (UPR). Human aortic endothelial cells (HAEC) were infected with Nox 5 or GFP adenovirus, RNA was extracted 12, 18 and 24 hours after infection and used to determine mRNA expression by a Gene Chip Human Transcriptome Array 2.0 (Affymetrix). Nox 5 overexpression after adenoviral infection in HAEC resulted in a significant enhancement of extracellular superoxide and hydrogen peroxide production and led to a significant increase in cellular apoptosis by caspases 3 and 7. The ontological analysis of the array showed an upregulation of UPR. Infection of HAEC with Nox 5 adenovirus activated the UPR pathway, increasing Ire1α and CHOP mRNA expression, GRP78 protein levels and reducing calnexin protein expression. Additionally, Nox 5 overexpression significantly reduced Nox 4 and NOS3 mRNA expression. Nox 5 overexpression promotes a situation of oxidative stress in HAEC, which alters correct protein folding and activates the UPR pathway.

ASPP2 Inhibits the Profibrotic Effects of Transforming Growth Factor-\u03b21 in Hepatic Stellate Cells by Reducing Autophagy

BackgroundApoptosis-stimulating protein of p53-2 (ASPP2) is a damage-inducible P53-binding protein that enhances damage-induced apoptosis. Fibrosis is a wound-healing response, and hepatic stellate cells (HSCs) are key players in liver fibrogenesis. However, little is known about the relationship between ASPP2 and hepatic fibrosis.AimsWe investigated the effects of ASPP2 overexpression in HSCs and the role of ASPP2 in mouse liver fibrogenesis.MethodsHuman HSCs (LX-2 cells) were pre-incubated with GFP adenovirus (Ad) or ASPP2 adenovirus (AdASPP2) for 24 h and then treated with or without TGF-β1. ASPP2+/− and ASPP2+/+ Balb/c mice were used to examine the effects of ASPP2 on liver fibrosis in vivo. ASPP2+/+ Balb/c mice were generated by injecting AdASPP2 into the tail vein of ASPP2 WT Balb/c mice; all mice received intraperitoneal injections of carbon tetrachloride.ResultsIn this study, ASPP2 was found to markedly inhibit TGF-β1-induced fibrogenic activation of LX-2 cells. Further experiments using an autophagic flux assay confirmed that ASPP2 reduced the fibrogenic activation of LX-2 cells by inhibiting autophagy. Moreover, we found that ASPP2 overexpression attenuated the anti-apoptotic effects of TGF-β1 in LX-2 cells. The extent of liver fibrosis was markedly reduced in ASPP2+/+ mouse liver tissue compared with control mice; however, in ASPP2+/− mice, hepatic collagen deposition was significantly increased.ConclusionThese results suggest that TGF-β1-induced autophagy is required for the fibrogenic response in LX-2 cells and that ASPP2 may both inhibit TGF-β1-induced autophagy and decrease liver fibrosis.

A Method Sustaining the Bioelectric, Biophysical, and Bioenergetic Function of Cultured Rabbit Atrial Cells.

Culturing atrial cells leads to a loss in their ability to be externally paced at physiological rates and to maintain their shape. We aim to develop a culture method that sustains the shape of atrial cells along with their biophysical and bioenergetic properties in response to physiological pacing. We hypothesize that adding 2,3-Butanedione 2-monoxime (BDM), which inhibits contraction during the culture period, will preserve these biophysical and bioenergetic properties. Rabbit atrial cells were maintained in culture for 24 h in a medium enriched with a myofilament contraction inhibitor, BDM. The morphology and volume of the cells, including their ability to contract in response to 1–3 Hz electrical pacing, was maintained at the same level as fresh cells. Importantly, the cells could be successfully infected with a GFP adenovirus. Action potentials, Ca2+ transients, and local Ca2+ spark parameters were similar in the cultured and in fresh cells. Finally, these cultured cells' flavoprotein autofluorescence was maintained at a constant level in response to electrical pacing, a response similar to that of fresh cells. Thus, eliminating contraction during the culture period preserves the bioelectric, biophysical and bioenergetic properties of rabbit atrial myocytes. This method therefore has the potential to further improve our understanding of energetic and biochemical regulation in the atria.

Fluorescence-guided surgery of a highly-metastatic variant of human triple-negative breast cancer targeted with a cancer-specific GFP adenovirus prevents recurrence

We have previously developed a genetically-engineered GFP-expressing telomerase-dependent adenovirus, OBP-401, which can selectively illuminate cancer cells. In the present report, we demonstrate that targeting a triple-negative high-invasive human breast cancer, orthotopically-growing in nude mice, with OBP-401 enables curative fluorescence-guided surgery (FGS). OBP-401 enabled complete resection and prevented local recurrence and greatly inhibited lymph-node metastasis due to the ability of the virus to selectively label and subsequently kill cancer cells. In contrast, residual breast cancer cells become more aggressive after bright (white)-light surgery (BLS). OBP-401-based FGS also improved the overall survival compared with conventional BLS. Thus, metastasis from a highly-aggressive triple-negative breast cancer can be prevented by FGS in a clinically-relevant mouse model.

Adenovirus-mediated transfer of the SOCS-1 gene to mouse lung confers protection against hyperoxic acute lung injury

Suppressor of cytokine signaling-1 (SOCS-1) is a member of the suppressor of cytokine signaling family of proteins and an inhibitor of interleukin-6 (IL-6) signaling. SOCS-1 has been shown to protect cells from cellular damage and apoptosis induced by tumor necrosis factor (TNF), lipopolysaccharide (LPS), and interferon gamma (IL-γ). However, it is not known whether increased SOCS-1 is protective during pulmonary oxidative stress. Therefore, we hypothesized that increased SOCS-1 in the lungs of mice would be protective in the setting of hyperoxic lung injury. We administered SOCS-1 adenovirus (Ad-SOCS-1) intratracheally into the lungs and exposed the mice to 100% O2. Mice infected with GFP adenovirus (Ad-GFP) were used as controls. Mice treated with Ad-SOCS-1 had enhanced survival in 100% oxygen compared to Ad-GFP-administered mice. After 3 days of hyperoxia, Ad-GFP mice were ill and tachypnic and died after 4 days. In contrast, all Ad-SOCS-1-treated mice survived for at least 6 days in hyperoxia and 80% survived beyond 7 days. Ad-SOCS-1 transfection protected mouse lungs from injury as indicated by lower lung wet/dry weight, alveolar–capillary protein leakage, reduced infiltration of inflammatory cells, and lower content of thiobarbituric acid-reactive substances in lung homogenate. Our results also indicated that Ad-SOCS-1 significantly inhibits hyperoxia-induced ASK-1 (apoptosis signal-regulating kinase 1) expression. Taken together, these findings show that increased expression of adenovirus-mediated SOCS-1 in the lungs of mice significantly protects against hyperoxic lung injury.

ASPP2 attenuates triglycerides to protect against hepatocyte injury by reducing autophagy in a cell and mouse model of non-alcoholic fatty liver disease.

ASPP2 is a pro-apoptotic member of the p53 binding protein family. ASPP2 has been shown to inhibit autophagy, which maintains energy balance in nutritional deprivation. We attempted to identify the role of ASPP2 in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In a NAFLD cell model, control treated and untreated HepG2 cells were pre-incubated with GFP-adenovirus (GFP-ad) for 12 hrs and then treated with oleic acid (OA) for 24 hrs. In the experimental groups, the HepG2 cells were pre-treated with ASPP2-adenovirus (ASPP2-ad) or ASPP2-siRNA for 12 hrs and then treated with OA for 24 hrs. BALB/c mice fed a methionine- and choline-deficient (MCD) diet were used to generate a mouse model of NAFLD. The mice with fatty livers in the control group were pre-treated with injections of GFP-ad for 10 days. In the experimental group, the mice that had been pre-treated with ASPP2-ad were fed an MCD diet for 10 days. ASPP2-ad or GFP-ad was administered once every 5 days. Liver tissue from fatty liver patients and healthy controls were used to analyse the role of ASPP2. Autophagy, apoptosis markers and lipid metabolism mediators, were assessed with confocal fluorescence microscopy, immunohistochemistry, western blot and biochemical assays. ASPP2 overexpression decreased the triglyceride content and inhibited autophagy and apoptosis in the HepG2 cells. ASPP2-ad administration suppressed the MCD diet-induced autophagy, steatosis and apoptosis and decreased the previously elevated alanine aminotransferase levels. In conclusion, ASPP2 may participate in the lipid metabolism of non-alcoholic steatohepatitis and attenuate liver failure.

Essential role of mTORC1 in the hypertension induced by overactivation of the brain renin‐angiotensin system (1067.2)

Angiotensin II (Ang II) is a major effector peptide of the renin‐angiotensin system (RAS). Ang II‐induced activation of angiotensin type 1 receptor (AT1R) signaling in cardiovascular‐related brain nuclei such as the subfornical organ (SFO) is thought to be important for the development of hypertension. However, the molecular machinery underlying the central pressor actions of Ang II is poorly defined. We previously demonstrated the involvement of neuronal mammalian target of rapamycin complex 1 (mTORC1) in blood pressure (BP) regulation. Interestingly, using in vitro systems we also found that mTORC1 is a downstream element of neuronal AT1R signaling. To assess the relevance of mTORC1 for Ang II action, we first examined mTORC1 activity in the cardiovascular brain regions in hypertensive transgenic mice with brain‐specific hyperactive RAS (sRA mice). Strikingly, the number of phospho‐S6 positive cells (used as a readout of mTORC1 activity) was significantly higher in the SFO of sRA mice (64±6) relative to controls (36±8, p<0.05, n=5). Moreover, central administration of an adenovirus encoding a dominant negative S6 kinase (an mTORC1 substrate) lowered systolic BP in sRA (from 148±8 to 130±8 mmHg, p<0.05, n=3) to comparable levels as control mice receiving GFP adenovirus (from 125±7 to 126±1 mmHg, n=3). These results indicate that neuronal mTORC1 activation is a critical event in brain RAS‐induced BP elevation.

ATP Synthase β-Chain Overexpression in SR-BI Knockout Mice Increases HDL Uptake and Reduces Plasma HDL Level.

HDL cholesterol is known to be inversely correlated with cardiovascular disease due to its diverse antiatherogenic functions. SR-BI mediates the selective uptake of HDL-C. SR-BI knockout diminishes but does not completely block the transport of HDL; other receptors may be involved. Ectopic ATP synthase β-chain in hepatocytes has been previously characterized as an apoA-I receptor, triggering HDL internalization. This study was undertaken to identify the overexpression of ectopic ATP synthase β-chain on DIL-HDL uptake in primary hepatocytes in vitro and on plasma HDL levels in SR-BI knockout mice. Human ATP synthase β-chain cDNA was delivered to the mouse liver by adenovirus and GFP adenovirus as control. The adenovirus-mediated overexpression of β-chain was identified at both mRNA and protein levels on mice liver and validated by its increasing of DiL-HDL uptake in primary hepatocytes. In response to hepatic overexpression of β-chain, plasma HDL-C levels and cholesterol were reduced in SR-BI knockout mice, compared with the control. The present data suggest that ATP synthase β-chain can serve as the endocytic receptor of HDL, and its overexpression can reduce plasma HDL-C.

The miR-143/145 Cluster Is a Novel Transcriptional Target of Jagged-1/Notch Signaling in Vascular Smooth Muscle Cells

Activation of Notch signaling by Jagged-1 (Jag-1) in vascular smooth muscle cells (VSMC) promotes a differentiated phenotype characterized by increased expression of contractile proteins. Recent studies show that microRNAs (miR)-143/145 regulates VSMC phenotype. The serum response factor (SRF)/myocardin complex binds to CArG sequences to activate miR-143/145 transcription, but no other regulators are known in VSMC. Using miR arrays, we found miR-143/145 induced following expression of a constitutively active Notch1 intracellular domain (N1ICD). We hypothesized that miR-143/145 is required for Jag-1/Notch-induced VSMC differentiation. Activation of Notch receptors by Jag-1 caused CBF1-dependent up-regulation of miR-143/145, increased differentiation, and decreased proliferation. Conversely, inhibiting basal Notch signaling decreased steady state levels of miR-143/145. Using SRF knockdown, we found that Jag-1/Notch induction of miR-143/145 is SRF independent, although full acquisition of contractile markers requires SRF. Using miR-143/145 promoter reporter constructs we show Jag-1/Notch increases promoter activity, and this is dependent on intact CBF1 consensus sites within the promoter. Chromatin immunoprecipitation (ChIP) assays revealed that N1ICD-containing complexes bind to CBF1 sites in the miR-143/145 promoter. We also identified N1ICD complex binding to CBF1 sites within the endogenous human miR-143/145 promoter. Using miR-143/145-interfering oligonucleotides, we demonstrate that Jag-1/Notch signaling requires induction of both miR-143 and miR-145 to promote the VSMC contractile phenotype. Thus, miR-143/145 is a novel transcriptional target of Jag-1/Notch signaling in VSMC. We propose miR-143/145 as activated independently by Jag-1/Notch and SRF in parallel pathways. Multiple pathways converging on miR-143/145 provides potential for fine-tuning or amplification of VSMC differentiation signals.

Biomechanical Regulation of Endothelium-dependent Events Critical for Adaptive Remodeling

Alterations in hemodynamic shear stress acting on the vascular endothelium are critical for adaptive arterial remodeling. The molecular mechanisms regulating this process, however, remain largely uncharacterized. Here, we sought to define the responses evoked in endothelial cells exposed to shear stress waveforms characteristic of coronary collateral vessels and the subsequent paracrine effects on smooth muscle cells. A lumped parameter model of the human coronary collateral circulation was used to simulate normal and adaptive remodeling coronary collateral shear stress waveforms. These waveforms were then applied to cultured human endothelial cells (EC), and the resulting differences in EC gene expression were assessed by genome-wide transcriptional profiling to identify genes distinctly regulated by collateral flow. Analysis of these transcriptional programs identified several genes to be differentially regulated by collateral flow, including genes important for endothelium-smooth muscle interactions. In particular, the transcription factor KLF2 was up-regulated by the adaptive remodeling coronary collateral waveform, and several of its downstream targets displayed the expected modulation, including the down-regulation of connective tissue growth factor. To assess the effect of endothelial KLF2 expression on smooth muscle cell migration, a three-dimensional microfluidic assay was developed. Using this three-dimensional system, we showed that KLF2-expressing EC co-cultured with SMC significantly reduce SMC migration compared with control EC and that this reduction can be rescued by the addition of exogenous connective tissue growth factor. Collectively, these results demonstrate that collateral flow evokes distinct EC gene expression profiles and functional phenotypes that subsequently influence vascular events important for adaptive remodeling.

STAT3 activation protects retinal ganglion cell layer neurons in response to stress

STAT3 is a major signaling molecule for many neurotrophic factors but its direct role in the protection of neurons in response to stress has not been addressed. We have studied the role of STAT3 in protecting retinal neurons from damage induced by ischemia/reperfusion and glutamate excitotoxicity by using adenovirus constructs to introduce active, normal or inactive STAT3 into retinal ganglion cells in culture and cells of the ganglion cell layer in the intact retina. Transient ischemia/reperfusion was induced in adult CD1 mice by elevating the intraocular pressure to the equivalent of 120 mmHg for 60 min, followed by a return to normal pressure. The levels, activation and distribution of STAT3 protein were evaluated by Western blot and immunocytochemistry. A transient peak of STAT3 activation was seen at 24 h post ischemia and a strong increase in STAT3 protein levels 24 h later. The increase in levels of STAT3 was detected in both ganglion cell bodies and processes in the plexiform layers by immunocytochemistry. The time course of STAT3 increase was slower than the time course of ganglion cell death as measured by TUNEL assay. Intravitreal injection of NMDA led to peak increases in activated STAT3 and STAT3 at 12 and 24 h post insult respectively. Purified RGCs were infected with recombinant wild-type STAT3, constitutively active and dominant negative forms of STAT3 adenoviruses or control empty virus and then treated with glutamate. Surviving infected cells were counted 24 and 48 h later. Infection with constitutively active STAT3 gave substantial protection when compared to the other constructs. Similarly, intravitreal injection of constitutively active STAT3 adenovirus one day before ischemia-reperfusion resulted in a decreased neural cell death in the ganglion cell layer compared with GFP adenovirus control. Our results suggest that persistent activation of STAT3 by neurotrophic factors provides strong neuroprotection and will be an effective strategy in a number of chronic retinal diseases.

Adenoviral Expression of I Ks Contributes to Wavebreak and Fibrillatory Conduction in Neonatal Rat Ventricular Cardiomyocyte Monolayers

Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K(+) current (I(Ks)) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of I(Ks) in rat cardiomyocyte monolayers contributes to wavebreak formation and facilitates fibrillatory conduction by promoting postrepolarization refractoriness. Optical mapping was performed in 44 rat ventricular myocyte monolayers infected with an adenovirus carrying the genomic sequences of KvLQT1 and minK (molecular correlates of I(Ks)) and 41 littermate controls infected with a GFP adenovirus. Repetitive bipolar stimulation was applied at increasing frequencies, starting at 1 Hz until loss of 1:1 capture or initiation of reentry. Action potential duration (APD) was significantly shorter in I(Ks)-infected monolayers than in controls at 1 to 3 Hz (P<0.05), whereas differences at higher pacing frequencies did not reach statistical significance. Stable rotors occurred in both groups, with significantly higher rotation frequencies, lower conduction velocities, and shorter action potentials in the I(Ks) group. Wavelengths in the latter were significantly shorter than in controls at all rotation frequencies. Wavebreaks leading to fibrillatory conduction occurred in 45% of the I(Ks) reentry episodes but in none of the controls. Moreover, the density of wavebreaks increased with time as long as a stable source sustained the fibrillatory activity. These results provide the first demonstration that I(Ks)-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.

Expression of Constitutively Active STAT3 Can Replicate the Cytokine-suppressive Activity of Interleukin-10 in Human Primary Macrophages

There is general agreement that signal transducer and activation of transcription 3 (STAT3) is required to mediate the anti-inflammatory activities of interleukin (IL)-10. However, STAT3 is activated by multiple factors that do not share the anti-inflammatory activity of IL-10. The question remains whether STAT3 is sufficient for the anti-inflammatory effects or whether there are other signals required, as had been suggested previously. We set out to map the human IL-10 receptor and to identify the key elements involved in transducing the cytokine-suppressive effects of IL-10. We were able to show an absolute requirement for both of the tyrosine residues found within the YXXQ-STAT3-docking site within the IL-10 receptor 1 and that no other signals appeared to be required. We used a constitutively active STAT3 to determine whether expression of this factor could suppress lipopolysaccharide-induced tumor necrosis factor and IL-6 production. Our data show that STAT3 activity can suppress both IL-6 and tumor necrosis factor production in lipopolysaccharide-stimulated macrophages. However, in synovial fibroblasts, STAT3 did not suppress IL-6 production, suggesting that the cellular environment plays an important role in dictating whether STAT3 drives a pro- or anti-inflammatory response.

CSX/Nkx2.5 Modulates Differentiation of Skeletal Myoblasts and Promotes Differentiation into Neuronal Cells in Vitro

CSX/Nkx2.5 transcription factor plays a pivotal role in cardiac development; however, its role in development and differentiation of other organs has not been investigated. In this study, we used C2C12 myoblasts and human fetal primary myoblasts to investigate the function of Nkx2.5 in skeletal myogenesis. The expression levels of Nkx2.5 decreased as C2C12 myoblasts elongated and fused to form myotubes. The expression of human NKX2.5 in C2C12 myoblasts inhibited myocyte differentiation and myotube formation, and up-regulated Gata4 and Tbx5 expression. The expression of NKX2.5 in terminally differentiated C2C12 myotubes resulted in a change in morphology and breakdown into smaller myotubes. Furthermore, overexpression of NKX2.5 in C2C12 cells and primary cultures of human fetal myoblasts led to differentiation of myoblasts into neuron-like cells and expression of neuronal markers. This study sheds light on the previously unknown non-cardiac functions of Nkx2.5 transcription factor.