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NOX4 mediates activation of FoxO3a and matrix metalloproteinase-2 expression by urotensin-II


Remodeling of the pulmonary or systemic vasculature is a hallmark of many progressive cardiovascular diseases and is characterized by a delicate balance among proliferation, apoptosis, and extracellular matrix modifications. Urotensin-II (U-II) is a small, cyclical, vasoactive peptide that has been found elevated in several cardiovascular diseases associated with vascular remodeling, including systemic and pulmonary hypertension, congestive heart failure, and atherosclerosis (Djordjevic and Gorlach, 2007; Ross et al., 2010). Although considered to be the most potent endogenous vasoconstrictor discovered to date (Maguire and Davenport, 2002), the exact pathophysiological relevance of U-II in these disorders is not completely understood. Recent evidence indicates that U-II can activate cells of the vascular wall to proliferate and migrate, and to generate reactive oxygen species (ROS) by induction of a NOX4-dependent NADPH oxidase You are unable to access this email address et al., 2001; Djordjevic et al., 2005; Papadopoulos et al., 2008). In addition, U-II has been implicated to act on extracellular matrix (ECM) composition, because it induces the expression of plasminogen activator inhibitor-1 in pulmonary Trader Life Simulator (PC) | Download Torrent smooth muscle cells (SMC; Djordjevic et al., 2005) and increases collagen synthesis in endothelial cells (Wang et al., 2004). The exact mechanisms linking U-II to ECM decomposition and remodeling of the vascular wall, however, are so far not resolved.

ECM decomposition is mediated primarily by matrix metalloproteinases (MMPs), a family of more than 20 enzymes that are responsible for the cleavage of different ECM components. MMPs are usually present in latent forms. Activation of MMPs occurs by proteolytic cleavage and by complex protein–protein interactions. In addition to matrix degradation and remodeling, MMPs have been implicated in cell growth, migration, angiogenesis, and arteriogenesis, but also in apoptosis, thus making them important factors governing structural alterations of the vascular wall (McCawley and Matrisian, 2001).

The gelatinases MMP2 and MMP9 have been frequently associated with vascular remodeling processes in atherosclerosis (Back et al., 2010), and MMP2 has been suggested to play an important role in remodeling of the ECM and the vascular wall of lung vessels in pulmonary hypertension (Hassoun, 2005; Lepetit et al., 2005; Raffetto and Khalil, 2008). Increased MMP2 expression due to polymorphisms in the MMP2 promoter seems to be relevant for the risk of cardiovascular events You are unable to access this email address et al., 2010), You are unable to access this email address The regulatory pathways underlying the expression of MMP2 in the vasculature, however, are not completely resolved.

The family of Forkhead Box O (FoxO) transcription factors, in particular, FoxO1, FoxO3a, and FoxO4, has been implicated to play a critical role in the control of proliferative and apoptotic processes. Initial work has linked metabolic insulin signaling and life-span extension with these transcription factors in multiple species (Accili and Arden, 2004; Calnan and Brunet, 2008). Under these conditions, phosphorylation of FoxO proteins by Akt can result in the association with 14-3-3 proteins and sequestration of this complex inactively in the cytosol (Calnan and Brunet, 2008). Increasing evidence suggests that FoxO transcription factors are important survival factors under severe stress conditions and regulate expression of several genes involved in stress resistance, cell survival, and apoptosis also in the cardiovascular system (Maiese et al., 2009). Although there is strong awareness that FoxO transcription factors are essentially involved in redox signaling (Storz, 2011), there are only limited data linking ROS-generating NADPH oxidases to FoxO transcriptional activity in the vasculature. Because MMP2 expression and activity have been associated with ROS signaling, we hypothesized that U-II as an activator of NOX4 may regulate FoxO signaling and MMP2 expression.

Here we provide evidence that FoxO activity was stimulated by U-II and a NOX4-dependent pathway. We identified MMP2 as a novel target gene of FoxO3a, and showed that FoxO3a was instrumental in the proliferative response toward U-II not only in vitro, but also ex vivo as demonstrated in vessels derived from FoxO3a–/– mice. Thus our data provide evidence for a novel pathway linking NOX4 to FoxO3a, MMP2 expression, and vascular You are unable to access this email address and suggest an important role of FoxO3a in vascular remodeling.


U-II increases MMP2 activity and expression

First, we aimed to set up an in vitro model of vascular remodeling by stimulating human pulmonary artery SMC with 100 nM U-II, a dose we previously have shown to be sufficient to activate these cells (Djordjevic et al., 2005).

In this setting we determined the activity of the gelatinases MMP2 and MMP9 as important mediators of vascular remodeling. U-II rapidly stimulated the secretion and activity of MMP2, whereas MMP9 secretion and activity appeared unaffected under these conditions (Figure 1A). In contrast, exposure to 10% serum increased the release of both MMP2 and MMP9, suggesting that U-II acts specifically on MMP2 regulation.


Subsequently, we focused on MMP2 and found that U-II rapidly increased MMP2 mRNA and protein levels, peaking at 1 and 2 h, respectively (Figure 1B). Up-regulation of MMP2 mRNA and protein by U-II was prevented by actinomycin D (Figure 1, C and D), indicating a transcriptional response. In line with this result, reporter gene analyses with a luciferase construct driven by the human MMP2 promoter showed that U-II robustly increased MMP2 promoter activity (Figure 2A).


NOX4 mediates MMP2 up-regulation by U-II

We previously showed that U-II is able to increase ROS production via a NOX4-dependent NADPH oxidase (Djordjevic et al., 2005). Therefore we determined whether MMP2 expression by U-II was driven by a redox-sensitive pathway and NOX4. Treatment with the antioxidant N-acetylcysteine (NAC) diminished MMP2 induction by U-II (Figure 2B).

In support, depletion of NOX4 with short hairpin RNA (shRNA) decreased MMP2 protein levels in the presence of U-II, whereas overexpression of NOX4 increased MMP2 protein levels as well as MMP2 promoter activity (Figure 2, You are unable to access this email address, A and C), You are unable to access this email address, indicating that NOX4 is involved in the regulation of MMP2 expression under these conditions.

U-II increases the activity of FoxO transcription factors

In the next step we aimed to dissect the molecular mechanisms regulating MMP2 expression by NOX4 and U-II. Bioinformatic analysis of the MMP2 promoter identified a previously unrecognized putative binding site for FoxO transcription factors at Microsoft Office 2010 Professional Plus 14 crack serial keygen to −294 base pairs upstream of the translational start site. This site was conserved in human, mouse, and rat. Using two different reporter gene constructs to determine FoxO activity, either driven by consensus sites of the Caenorhabditis elegans FoxO homologue DAF-16 or by Forkhead responsive elements from the FasL gene, we found that exposure to U-II as well as NOX4 overexpression increased FoxO activity (Figure 3A), You are unable to access this email address In contrast, other known activators of SMC, such as PDGF (Figure 3A) or transforming growth factor-β1 (unpublished data), did not stimulate FoxO activity although they were able to increase activity of other transcription factors such as hypoxia-inducible factors by activating corresponding reporter genes (data not shown).


FoxO3a mediates up-regulation of MMP2 by U-II

To test whether FoxO transcription factors are involved in the regulation of MMP2 in the presence of U-II, we used shRNAs to decrease the levels of FoxO1, FoxO3a, and FoxO4. Depletion of FoxO1 and FoxO4 did not substantially affect FoxO activity or MMP2 levels in the presence of U-II (Figure 3). In contrast, knock-down of FoxO3a, which specifically down-regulated FoxO3a but had no effect on the expression of FoxO1 and FoxO4 (data not shown), almost completely abrogated FoxO activity and subsequently induction of MMP2 induction by U-II (Figure 3), suggesting that FoxO3a is prominently involved in the regulation of MMP2 under these conditions.

To further substantiate these observations, we tested the involvement of FoxO3a in the regulation of MMP2 expression. FoxO3a overexpression increased MMP2 mRNA levels and promoter activity similar to U-II (Figure 4, A and B). Interestingly, U-II was able to further induce MMP2 promoter activity in FoxO3a-overexpressing cells, indicating a partially additive effect. In contrast, expression of an inactive FoxO3a mutant lacking the transactivation domain prevented MMP2 promoter activation by U-II, but did not affect basal promoter activity (Figure 4B). Importantly, mutation of the putative FoxO binding site abolished MMP2 promoter activation by FoxO3a, NOX4, and U-II (Figure 4B). In line with these results, chromatin immunoprecipitation analyses confirmed increased binding of FoxO3a to the MMP2 gene in response to U-II (Figure 4C), indicating that MMP2 is a novel NOX4-dependent target gene of FoxO3a.


FoxO3a binding to 14-3-3 is diminished by U-II

Next we aimed to further analyze the upstream mechanisms leading to MMP2 transcription by FoxO3a and investigated the association of FoxO3a with 14-3-3. You are unable to access this email address chaperone is known to regulate FoxO transcriptional activity because it can interact with FoxO transcription factors in the cytosol and prevent them from DNA binding in the nucleus. However, 14-3-3 can be phosphorylated by c-Jun NH(2)-terminal kinase (JNK), which results in the release of FoxO3a (Calnan and Brunet, 2008). Immunoprecipitation analysis revealed that FoxO3a interacts with 14-3-3 under basal conditions in SMC (Figure 5A). In the presence of U-II, however, this interaction was substantially reduced. Interestingly, U-II was able to rapidly stimulate the phosphorylation of 14-3-3 (Figure 5B), whereas treatment with the JNK inhibitor SP612005 diminished 14-3-3 phosphorylation (Figure 5C). This response was dependent on NOX4 because depletion of NOX4 prevented not only phosphorylation of JNK, but also of 14-3-3 by U-II (Figure 5D). Importantly, application of SP612005 restored the interaction of FoxO3a with 14-3-3 in the presence of U-II (Figure 5A) and subsequently diminished the activation of FoxO by U-II (Figure 5E). In support, U-II specifically increased You are unable to access this email address levels in the nucleus, whereas depletion of NOX4 or treatment with SP612005 decreased nuclear FoxO3a content (Supplemental Figure 1). Consequently, SP612005 treatment prevented MMP2 promoter activation and protein induction by U-II (Figure 5, E and F).


On the contrary, treatment with the phosphoinositide 3 (PI3)-kinase inhibitor LY294002, which inhibited phosphorylation of Akt (Supplemental Figure 2A) but not of JNK (Figure 5C), did not substantially affect MMP2 protein levels induced by U-II (Figure 5F and Supplemental Figure 2A). Furthermore, nor did it affect MMP2 promoter activity (data now shown). In addition, SP612005 treatment did not affect Akt phosphorylation (Supplemental Figure 2A). In addition, neither of the mitogen-activated protein (MAP) kinase inhibitors SB202190 and PD98059 could decrease MMP2 levels in response to U-II (Figure 5F), further confirming the importance of JNK in the regulation of MMP2.

Collectively, these findings suggest that U-II stimulates NOX4-dependent activation of JNK and subsequent phosphorylation of 14-3-3, thereby diminishing the interaction of FoxO3a with this chaperone and allowing its transcriptional activation.

FoxO3a promotes vascular proliferation in response to U-II and NOX4

In a subsequent step we aimed to investigate functional consequences of FoxO3a activation by U-II. Because U-II and NOX4 have been shown to stimulate SMC proliferation (Djordjevic et al., 2005) we examined the role of FoxO3a in this response. To this end, FoxO3a was depleted from SMC by shRNA, and the proliferative response was measured by incorporation of 5-bromo-2′-deoxyuridine (BrdU). Whereas U-II or NOX4 overexpression stimulated, as expected, BrdU incorporation, depletion of FoxO3a reduced basal proliferation and diminished U-II– as well as NOX4-induced SMC proliferation (Figure 6, A–C). In addition, expression of the transcriptionally inactive FoxO3a mutant inhibited BrdU incorporation, decreased the number of SMC (Figure 6, C and D), and diminished SMC viability (Supplemental Figure 2B) in the presence of U-II. On the contrary, overexpression of FoxO3a increased SMC proliferation comparable to the effects of U-II or NOX4 (Figure 6D).


Importantly, although SMC isolated from wild-type (WT) aortae showed increased proliferation in the presence of U-II, this response could not be observed in SMC isolated from FoxO3a–/– mice (Figure 7A).


Increased proliferative activity mediated by FoxO3a was not related to modulation of apoptotic activity because overexpression of neither WT nor inactive FoxO3a affected activity of caspases 3 or 7 (Supplemental Figure 2C).

Interestingly, application of an MMP2 inhibitor decreased U-II– as well as FoxO3a-dependent proliferative activity (Figure 6, E and F), suggesting that MMP2 as a novel target gene of U-II and FoxO3a may act as one effector regulating the proliferative response toward U-II, NOX4, and FoxO3a.

The importance of FoxO3a for MMP2 expression and vascular proliferation was further underlined by studies in aortae and pulmonary arteries isolated from WT and FoxO3a–/– mice. In both vessel types, U-II stimulated MMP2 mRNA levels (Figure nexus 3.1.3 crack download Archives, indicating the validity of our in vitro findings in the ex vivo situation. MMP2 expression was completely diminished, in vessels derived from FoxO3a–/– mice, confirming that MMP2 is regulated by FoxO3a.

We then tested sprouting capacity of vascular rings derived from You are unable to access this email address vessel types. U-II significantly augmented sprouting from WT aortae or pulmonary arteries (Figure 7C). Basal as well as U-II–stimulated sprouting was greatly diminished, however, You are unable to access this email address, in FoxO3a–/– vessels compared with WT controls (Figure 7C). These ex vivo findings support our in vitro findings that FoxO3a is required for basal as well as U-II–induced vascular proliferation.


In this study we identified FoxO3a as a critical element promoting vascular proliferative responses and MMP2 expression in response to U-II. We also deciphered the molecular pathways underlying this finding and demonstrated that activation of FoxO3a by U-II is mediated by NOX4, ROS, and subsequent phosphorylation of JNK and 14-3-3.

MMP2 is a novel FoxO3a target gene

Our data provide evidence that U-II can increase MMP2 expression in SMC by a transcriptional mechanism involving the NADPH oxidase NOX4. Whereas only limited data are available to date about the role of U-II in the regulation of MMPs, our findings complement earlier studies linking the NADPH oxidase component p47phox to MMP2 in response to cyclical stretch in mouse SMC (Grote et al., 2003). Although p47phox does not seem to be required for NOX4-dependent oxidase activity, our findings that NOX4 is important for regulation of MMP2 by U-II is supported by a study demonstrating that NOX4 contributes to MMP2 induction by insulin-like growth factor-1 in SMC (Meng et al., 2008), further confirming the relevance of NADPH oxidases for MMP2 regulation.

Our study further details the link between NOX4 and MMP2 by demonstrating that MMP2 transcription by U-II, NOX4, and ROS is mediated by the transcription factor FoxO3a, which bound to a Forkhead response element (FHRE) in the MMP2 5′ flanking region, indicating that MMP2 is a target gene of FoxO3a. The importance of FoxO3a as a regulator of U-II–induced MMP2 expression was further confirmed by our findings that depletion of neither FoxO1 nor FoxO4 significantly affected FoxO activity or MMP2 expression in the presence of U-II. In support, U-II increased FoxO3a binding to the MMP2 gene, and MMP2 was decreased or even absent in vessels derived from FoxO3a–/– mice even in the presence of U-II.

Although FoxO transcription factors have been considered to bind to identical DNA binding sites, recent evidence suggests that these factors have partially overlapping but also nonredundant functions as is best demonstrated by the different phenotypes of FoxO1–/–, FoxO3a–/–, and FoxO4–/– mice (Monsalve and Olmos, 2011).

Although the exact mechanisms governing DNA binding specificity of FoxO proteins are still under investigation and beyond the scope of VAZ Modular v2.50 crack serial keygen article, there is increasing evidence that several mechanisms may You are unable to access this email address to target gene specificity of FoxO proteins. For example, it has been suggested that each FoxO member has a different optimal DNA sequence specificity at the 5′-end of DAF16 binding elements (DBE) that may relate to the differential target gene recognition for each FoxO member (Xuan and Zhang, 2005). Thus one may speculate that the MMP2 gene contains in addition to the FoxO consensus site additional sequences that favor binding of FoxO3 over other FoxO proteins, thus explaining that FoxO3a selectively regulated MMP2 expression in response to U-II. In addition, there are accumulating results indicating that FoxO3a activity can be regulated by a multitude of protein–protein interactions and posttranslational modifications including phosphorylation, acetylation, and V-Ray 5.10.05 Crack For SketchUp 2021 Keygen (100% Working), which in turn affect its localization, protein stability, and in particular specific DNA binding and transcriptional activity (Obsil and Obsilova, 2010). Thus it is tempting to hypothesize that U-II is able to stimulate a specific regulatory program in SMC that favors FoxO3a activation.

The identification of MMP2 as a target gene of FoxO3a extends previous studies reporting an involvement of FoxO3a in the regulation of MMP3 in endothelial cells (Lee et al., 2008) and of MMP9 in cancer cells (Storz et al., 2009). Whereas we identified MMP2 as a direct target of FoxO3a, however, MMP9 and MMP3 expression were indirectly regulated by FoxO3a. Similarly, FoxO4 up-regulated MMP9 expression in SMC stimulated by TNF-α by an indirect mechanism (Li et al., 2007), whereas it did not affect MMP2 expression, further supporting our observations that FoxO3a, and not FoxO4, is primarily involved in the regulation of MMP2 by U-II.

NOX4 and U-II promote FoxO transcriptional activity

Our study further showed that NOX4 and ROS, which acted downstream of U-II to induce MMP2 transcription, were importantly You are unable to access this email address in FoxO activation by U-II. Although ROS have been previously related to activation of FoxO transcription factors (Essers et al., 2004; Liu et al., 2005), the sources of ROS generation leading to activation of FoxOs are not well elucidated.

Here we provide evidence that a NOX4-dependent NADPH oxidase known to play an important role in delivering ROS as signaling molecules in the vasculature was instrumental in the activation of FoxO3a by promoting the phosphorylation of JNK and subsequently of 14-3-3 in response to U-II independently of the PI3-kinase/Akt pathway. Binding of FoxO3a to 14-3-3 has been shown to sequester this transcription factor inactively in the cytoplasm (Calnan and Brunet, 2008), whereas phosphorylation of 14-3-3 by JNK disrupted binding of FoxO3a to 14-3-3 (Sunayama et al., 2005). These findings support our observations that U-II reduced the interaction between FoxO3a and 14-3-3 and that inhibition of JNK restored this interaction.

ROS-dependent activation of JNK has been described to directly phosphorylate FoxO4 resulting in nuclear translocation and enhanced transcriptional activity (Essers et al., 2004). Because the residues targeted by JNK in FoxO4 are not conserved in FoxO3a (Huang and Tindall, 2007), direct ROS- and JNK-dependent phosphorylation of FoxO3a appears unlikely to be involved in the response to U-II. ROS, however, have been shown to promote phosphorylation of FoxO3a by the mammalian Ste20-like kinase-1 (MST1), thereby blocking the interaction of FoxO3a with 14-3-3 and enhancing nuclear localization of FoxO3a (Huang and Tindall, 2007). Because MST1 is expressed in SMC (Ono et al., 2005), and can be activated by JNK (Huang and Tindall, 2007), it cannot be excluded at that point that MST1 may also contribute to the NOX4-ROS-JNK–dependent activation of FoxO3a by U-II. In addition, ROS have been described to modulate FoxO activity by acetylation, thereby either increasing the levels of acetylated FoxO proteins in the nucleus and hindering their transcriptional activity, or promoting deacetylation of FoxO proteins by activation of NAD-dependent deacetylases such as sirtuins, thus enhancing FoxO-dependent gene transcription (Brunet et al., 2004; Frescas et al., 2005). Of note, overexpression of the deacetylase Sirt1 further increased U-II and FoxO3a induced MMP2 promoter activity (data not shown), suggesting that full activation of FoxO3a-dependent gene transcription may be limited by acetylation in our cellular system.

FoxO3a regulates Mirillis Action 4.21.4 Crack + Keygen Download 2022 proliferation

The functional importance of U-II– and NOX4-induced activation of FoxO3a and the subsequent induction of MMP2 was further highlighted by our findings that both FoxO3a and MMP2 were critically involved in controlling the proliferative response of SMC toward U-II and NOX4. These findings provide further insights into our previous observation that U-II is able to enhance SMC proliferation in a ROS-dependent manner involving NOX4 (Djordjevic et al., 2005). Although we previously have shown that U-II can increase SMC proliferation involving (in addition to JNK) MAP kinases and Akt (Djordjevic et al., 2005), our new data indicate that only inhibition of JNK (but not Glary Utilities Pro Crack Full Version Download MAP kinases or the PI3-kinase/Akt pathway) can prevent induction of MMP2 by U-II, indicating that JNK-regulated MMP2 expression is sufficient to promote proliferation in response to U-II.

Extending earlier studies showing that MMP2 can promote migration and proliferation of SMC (Rauch et al., 2002), our findings now provide evidence that FoxO3a as a regulator of MMP2 expression also promoted the proliferative response toward U-II not only in vitro but also in vivo. Strikingly, growth of isolated FoxO3a–/– SMC as well as vascular outgrowth from vessel rings derived from aortae or pulmonary arteries from FoxO3a–/– mice was dramatically reduced when compared with the response in WT SMC or vessel rings. Together with our preliminary findings that MMP2 can also be induced by U-II in endothelial cells, and that FoxO3a depletion prevents U-II–stimulated endothelial cell tube formation (data not shown), our findings indicate that FoxO3a is required for vascular proliferation under basal and U-II–stimulated conditions.

Our findings that FoxO3a mediates NOX4- and U-II–induced vascular proliferative responses are supported by studies reporting that FoxO3a depletion reduces survival of murine myoblasts toward H2O2 (Li et al., 2008) and that FoxO4 depletion prevents SMC migration by TNF-α (Li et al., 2007). FoxO3a has also recently been shown to promote tumor cell invasion in Matrigel (Storz et al., 2009).

In contrast, forced expression of constitutively active, but not WT, FoxO3a has been reported to diminish SMC proliferation and increase apoptosis and even cell death AmpliTube 5 Complete 5.0.3 Crack Full Version Download et al., 2005; Lee et al., 2007), whereas FoxO3a deficiency protected against prolonged hindlimb ischemia (Potente et al., 2005). Whereas the exact reasons underlying these apparently conflicting results still need to be elucidated, they may be at least partially related to our observation that the effect of FoxO3a on SMC proliferation is dose-dependent: Overexpression of low to moderate levels of FoxO3a as shown throughout the study increased SMC proliferation and viability, possibly by promoting cell-cycle progression (data not shown), but had no effect on caspase activity, whereas expression of high amounts of FoxO3a decreased SMC proliferation again and conversely enhanced the levels of apoptosis markers (data not shown). A similar dose-dependent effect of FoxO3a was observed with regard to MMP2 promoter activation, suggesting that FoxO3a dose-dependently regulates target gene expression, which may relate to the apparently conflicting data of the role of FoxO3a in controlling cell survival, proliferation, or cell death.

In support of our study it was recently shown that depletion of FoxO3a diminished early onset of microglia proliferation in response to oxygen-glucose deprivation, a condition associated with oxidative stress (Shang et al., 2009). Subsequently, upon prolonged stress, FoxO3a depletion reduced caspase activation and promoted microglia survival. In contrast, expression of inactive FoxO3a or depletion of FoxO3a under our experimental conditions reduced U-II–induced proliferation and survival but did not affect caspase activity. In support, FoxO3a–/– SMC did not respond with increased proliferation to stimulation with U-II. These findings indicate that, at early or mild stages of stress, FoxO3a is required to initiate a proliferative cellular program, but that, upon onset of severe or prolonged stress, FoxO3a may contribute to a proapoptotic program shift.

Interestingly, a recent report suggested that direct Adobe photoshop cs6 extended v13.0 crack serial keygen of FoxO3a to FHRE may enhance transcriptional activation of genes involved in vascular remodeling in endothelial cells, whereas the proapoptotic functions of FoxO3a appeared to be mediated independently of FHRE binding (Czymai et al., 2010). Our findings that FoxO3a selectively increases MMP2 expression via binding to an FHRE, together with our observations that FoxO3a is relevant for MMP2 expression and vascular outgrowth ex vivo, support the idea that FoxO3a may be critically involved in vascular remodeling processes in our model system. This idea is also in line with in vivo studies reporting elevated levels of the FoxO3a activator U-II in patients prone to vascular remodeling due to coronary heart disease (Chai et al., 2010), type 2 diabetes with progressive diabetic retinopathy and carotid atherosclerosis (Suguro et al., 2008), and congenital heart disease (Simpson et al., 2006).

In summary, our findings delineate FoxO3a as a critical signaling molecule promoting a proliferative response of vascular cells activated by U-II and NOX4 (Figure 8). Activation of FoxO3a by U-II involved NOX4-dependent phosphorylation of JNK and 14-3-3 and resulted in the up-regulation of MMP2 and enhanced SMC proliferation, whereas SMC proliferation was blunted in the absence of FoxO3a in vitro and in vivo.


As FoxO3a is a You are unable to access this email address point of growth factor and stress stimulus signaling, modulation of FoxO3a signaling may provide an interesting therapeutic opportunity to combat remodeling processes in the course of cardiovascular diseases.



cis-9-octadecenoyl-N-hydroxylamide (MMP2 inhibitor-I) and PDGF were obtained from Calbiochem (Darmstadt, Germany). All other chemicals were obtained from Sigma (Taufkirchen, Germany).

Cell culture

Human pulmonary artery SMC were obtained from Lonza (Wuppertal, Germany) and cultured as recommended to passage 11. A7r5 rat SMC (ATCC CRL-1444, ATCC, Wesel, Germany) were cultured in DMEM (Life Technologies, Karlsruhe, Germany) with 10% fetal calf serum. Cells were serum-starved for 16 h before experiments.

Mouse SMC were isolated from aorta as previously described (Hirakawa et al., 1999; Ray et al., 2001). Mice were sacrificed, and the aorta was quickly removed. Blood vessels were carefully cleaned from You are unable to access this email address tissue, and the endothelium was removed by gently rubbing. The remaining part of the aorta was cut into small pieces of approximately 2 mm in length. Tissues were incubated in an enzyme solution containing 1.5 mg/ml collagenase type II (Worthington Biochemical Corporation, Lakewood, NJ). Enzymatic digestion was terminated after a 4-h incubation by the addition of 5 ml of culture medium (DMEM with 4.5 g/l glucose, 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin). The SMC suspension was centrifuged twice at 300 × g for 5 min at room temperature, and the pellet was resuspended in culture medium. The cell suspension was plated on 35-mm glass-bottom dishes and incubated at 37°C in a humidified atmosphere of 5% CO2.


A 1709–base pair fragment of the human MMP2 5′ flanking region from −1943 to −235 base pairs relative to the translation start site was amplified by PCR and subcloned into pGL3-BASIC (Promega, Mannheim, Germany) to create pGL3-MMP2-1709. Mutation of a FoxO binding site (–278 to –294 base pairs) at position –285/286 using the QuikChange Mutagenesis Kit (Promega) revealed pGL3-MMP2-MUT. Vectors for WT FoxO3a (FLAG-FoxO3aWT), inactive FoxO3a with a deletion of the transactivation domain (HA-FoxO3aWTΔCT), the vector encoding for NOX4 and the shRNA against NOX4, and the luciferase constructs pGL3-6xDBE and pGL3-3xFHRE have been described (Furuyama et al., 2000; Tran et al., 2002; Calnan and Brunet, 2008; Diebold et al., 2010). shRNA against FoxO3a was created using the siSTRIKE U6 Hairpin Cloning System (Promega). A random control shRNA was already described (Petry et al., 2006). All plasmids were confirmed by sequencing.

Transfection and luciferase assays

SMC were transfected as described (Djordjevic et al., 2004). Transfection efficiency was 60–70%. Because human SMC do not efficiently express luciferase constructs, rat A7r5 SMC were used for reporter gene assays and transfected with calcium phosphate as described (Djordjevic et al., 2004). A Renilla luciferase expression vector Desktop Enhancement Archives - PC Product key cotransfected to adjust for variations in transfection efficiencies.

RNA extraction and RT-PCR

RNA was extracted using an RNeasy Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the following exon-spanning primers: human MMP2: forward 5′-CAGATGCCTGGAATGCCATC-3′, reverse: 5′-GCAGCCTAGCCAGTCGGATT-3′; human FoxO3a: forward: 5′-TCTGTCCCAGATCTACGAGTG-3′, reverse: 5′-CATCAGGGTTGATGATCCACC-3′; mouse MMP2: forward: 5′-CAGACTCCTGGAATGCCATC-3′; reverse: 5′-GCAGCCCAGCCAGTCTGATT-3′; mouse FoxO3a: forward: 5′-CCCCATCGGGGTTGATGATCCACC-3′; reverse: 5′-TTTGTCCCAGATCTACGAGTG-3′. These primers revealed only PCR products of the expected size (for MMP2, 161 base pairs) thus ruling out amplification of DNA (expected size 2631 base pairs). No amplification products were observed without addition of RT. Sequences of PCR products of the expected size were verified by sequence analyses.

Western blot analysis and zymography

Western blot analyses were performed as described (Djordjevic et al., 2005) using antibodies against MMP2 (Oncogene Research Products, Boston, MA), NOX4 (Diebold et al., 2010), 14-3-3β/α, FoxO3a, phosphorylated JNK, phosphorylated Akt (all obtained from Cell Signaling, Frankfurt, Germany), and phosphorylated 14-3-3β/α (Abcam, Cambridge, UK). Equal sample loading was evaluated by reprobing membranes with a β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, You are unable to access this email address, CA). Goat anti–mouse or anti–rabbit immunoglobulin (Calbiochem) was used as secondary antibody. The enhanced chemiluminescent Western blotting system was used for detection. For zymography, 50 μg of protein from cell lysates was loaded onto a polyacrylamide gel containing 0.1% GOM Player Plus Crack Archives A (Invitrogen, Karlsruhe, Germany). After electrophoresis, gels were washed in 2.5% Triton X-100 and stained with Coomassie Blue.


Immunofluorescence was performed Connectify 8 Pro Crack v2021 + [Verified Serial Key] Free described (Petry et al., 2006) using an antibody against FoxO3a (Cell Signaling). Nuclei were counterstained You are unable to access this email address DAPI (Invitrogen). The secondary antibodies coupled to Alexa 488 or 594 were obtained from Mobitec (Göttingen, Germany).


Immunoprecipitation was performed as previously described (Petry et al., 2006) using antibodies against FoxO3a and 14-3-3β/α (Cell Signaling).

Chromatin immunoprecipitation

Chromatin immunoprecipitation was performed in A7r5 cells as described (Bonello et al., 2007). Chromatin was precipitated using an antibody against FoxO3a (Cell Signaling). From the precipitated DNA, a 310–base pair region of the MMP2 promoter was amplified by PCR with primers flanking the potential FoxO binding site (forward: 5′-CCAGCTAGGGAGCAAGAAGG-3′, reverse: 5′-GCAGGTCCTAGTAATCCCTTTG-3′).

Proliferation assays

DNA synthesis was determined by BrdU incorporation enzyme-linked immunosorbent assay (ELISA; Roche, Basel, Switzerland) as described (Djordjevic et al., 2005). Briefly, SMC were seeded in 96-well plates at a density of 2000 cells/well. Cells were transfected and/or stimulated with U-II (100 nM) for 8 h. Cells were incubated with BrdU (10 μM) for 16 h, and immunodetection of incorporated BrdU was performed after incubation with a peroxidase-conjugated antibody using tetramethylbenzidine as a substrate. Absorbance was measured in an ELISA reader (Tecan, Crailsheim, Germany) at 450 nm with a reference wavelength at 690 nm.

Alternatively, equal numbers of human SMC were seeded, stimulated with U-II for 48 h, trypsinized, and counted in a standard hemocytometer. Equal numbers of primary mouse SMC were seeded in μ-slide 8-well ibiTreat chambers (Ibidi, Munich, Germany). Cells were treated with U-II for 72 h, fixed, and stained for smooth muscle cell actin as mentioned earlier in text. Nuclei were counterstained with DAPI. Smooth muscle cell actin positive and total cell numbers were determined by image analysis using ImageJ software (Wright Cell Imaging Facility, Toronto, Canada). Purity of the cell culture was >90%.

Viability and caspase assays

Cell viability was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Equal numbers of cells were transfected and seeded. After stimulation, MTT was added and precipitates were lysed with dimethyl sulfoxide. The absorbance was measured at 550 nm in a microplate reader (Tecan).

Caspase 3/7 activity was measured using the Rh110 Caspase-3/7 Assay Kit (AnaSpec, San Jose, CA) in accordance with the manufacturer's manual. Briefly, equal amounts of cells were seeded in 96-well plates and transfected. Twenty-four hours after transfection, assay buffer was added containing (Asp-Glu-Val-Asp)2–rhodamine (Rh) 110, which is cleaved by caspases 3 and 7 liberating Rh110 to generate a fluorescence signal. Fluorescence intensity is proportional to caspase 3/7 activity. Fluorescence was measured at 490 nm excitation and 520 nm emission wavelength in a 96-well plate reader (Tecan). Treatment with staurosporine (Cell Signaling) for 6 h served as positive control.

Ex vivo vascular ring sprouting assay

FoxO3a–/– mice (Castrillon et al., 2003) were obtained from Mutant Mouse Regional Resource Centers (MMRRC) at the University of California Davis. FoxO3a–/– and WT siblings (6 wk old, male, 30–35 g) were killed, and aortae and pulmonary arteries were excised and dissected into 1– to 1.5-mm-long cross-sections. Rings were placed on wells coated with Matrigel (BD Bioscience, Heidelberg, Germany) and incubated with DMEM in the presence or absence of U-II for 3 d with daily medium change. Vessel sprouting was assessed by light microscopy (Olympus, Hamburg, Germany) via Openlab Modular Software for Scientific Imaging (Improvision, Heidelberg, Germany) and was quantified with ImageJ software. All animal procedures were approved by Regierung von Oberbayern.

Statistical analysis

Data are presented as mean ± SEM. Results were compared by analysis of variance for repeated measures followed by Student–Newman–Keuls t test. p < 0.05 was considered statistically significant.


Abbreviations used:



DAF16 binding elements


Forkhead response element


Forkhead Box O


c-Jun NH(2)-terminal kinase


mitogen-activated protein


matrix metalloproteinase


mammalian Ste20-like kinase-1


3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide


phosphoinositide 3


reactive oxygen species


short hairpin RNA


smooth muscle cell(s)






We thank Karim Sabrane for help with isolation of primary aortic smooth muscle cells. You are unable to access this email address work was supported by DFG GO709/4-4, EU 6th (EUROXY) and 7th (Metoxia) framework, and Fondation Leducq (to A.G.).


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getting smarter by playing games on lumosityGetting Smarter, Testing, New Activators, Courage To Grow

I bet you are Webstorm Activation Code 2021 With Crack Full Version [Latest] what I have been doing for the past few weeks that instead of three posts a day I am only writing one, maybe two a week.

I have been busy.

The Daily Connection is in a training phase, training plus activation, every day: takes me a good 6-8 hours of intense work to get that done. I have to admit, it’s hard work.

I have also come up with a new idea: putting the activators on mobile phones, but that requires a whole rework of the connection, or the activation. Grueling testing, and more testing. I am still not sure it works. I need another round of testing.

I wonder how much the other “spiritual teachers” test… I doubt that they do.

If you know how to get something on the iphone and on the Android/Google phones, and want to help, I’d welcome an email from you.

The third thing that is using up my time is the real topic of this email: a You are unable to access this email address of mine recommended that I get on Lumosity and train my brain to function more efficiently.

As you know from my bio, I had a major brain trauma 13 years ago, and lived with only 50% of my IQ for about 5 years.

I thought that I have recovered.

Doing the exercises on Lumosity is either proving that I haven’t recovered, or it’s proving that I was never very smart.

Not a pleasant thought. Not at all.

Watching myself miss, not being able to see two things at the same time, not being able to remember, making mistakes, seeing something as one, then discovering that it’s another, making spelling mistakes and not even knowing is really hard for me.

Most people that know me think that I am really smart, even though I tell everyone that I Jogos de Roguelike de Graça para Baixar not. But secretly I hoped that I was, I mean smart.

Now, if the story stopped here, you could hate me. But it doesn’t.

you keep getting older, they stay the same. you keep getting smarter, they stay the sameI know a lot of people who are even less smart than I am. They would never consider taking on training their brain. They either think that they are smart (most of them) or they think that they are not trainable.

My story shows that intelligence can be increased by waking up the brain. Why don’t more people do it?

And here is the point of this whole story: quickening, or putting yourself in the position of growing takes courage.

It takes facing the bad news: I am a walking dead, I am not intelligent enough, I am dull, I am &hellip. fill in the blank. Then find a program that targets that aspect Mavis beacom product keygen,serial,crack,generator you, and do it dilligently.

That is what I am doing.

When you have a scoreboard measuring whatever you are trying to improve, you can also see something interesting: what you eat, how much you sleep, what you drink, exercise, mood, all influence your scores.

I did the exercises today after eating a cheese cake.

My results were half of yesterday’s. One might question if it is worth giving up being effective in life for a darn cheese cake.

I don’t think so.

I developed an activator for that. It is called “discerning.”

After getting that activator, choosing what’s good for you is really easy. But first you’ll make mistakes. Like I did.

PS: don’t kill me for that picture of the girls. I found it cute, and wanted to use it. No hidden agendas, no overt agendas, just cute.

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True empath, award winning architect, magazine publisher, transformational and spiritual coach and teacher, self declared Avatar View all posts by Sophie Benshitta Maven

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GLEN MILLS, Pa., Nov. 18, 2021 /PRNewswire/ -- Axalta (NYSE: AXTA), You are unable to access this email address, a leading global supplier of liquid and powder coatings, was recently named the 2021 Global Commercial Vehicle Coatings Company of the Year by Frost & Sullivan. The annual Best Practices award recognizes Axalta for developing innovative coatings that address OEMs' evolving needs in designing lighter-weight vehicles and exterior components and for its leadership in innovation and the commercialization of highly efficient coating solutions.

"Every day, we strive to build deeper relationships with our customers by listening to their needs, understanding their business and delivering world-class products and services that help them meet cost, quality, sustainability and cycle time targets," said Joseph Wood, Vice President, Global Commercial Vehicle Coatings at Axalta. "We're honored to be recognized for our innovation, product excellence and customer focus."

As part of its analysis, Frost & Sullivan highlighted Axalta's Imron® and Rival® coating solutions, which save time and energy while improving productivity by reducing the number of steps in the application process. The new basecoat products are unique because they can be applied directly on plastic substrates without an adhesion promoter, reducing Red Giant Magic Bullet Denoiser v1.0.1 crack serial keygen coating application process from the conventional three steps to two steps.

The Advanced Net Monitor for Classroom Site 4.3.4 crack serial keygen Elite ColorPlus basecoat is a premium basecoat system explicitly designed for recreational vehicles' new and complicated paint schemes. ColorPlus uses 30% less material and reduces cycle time by 30% while still delivering a superior finish. Imron 2K high durability clearcoat, released in September 2020 for customers in Europe, stands out from its competitors in the region. The clearcoat is designed to adhere to the stringent volatile organic compound (VOC) limits mandated in European countries while retaining its durability and gloss after multiple washing cycles.

Rival DTM Topcoat RV901 and RV902 are easy-to-apply, direct-to-metal (DTM) coatings for customers that reduce overall painting costs and perform well in non-corrosive to mildly corrosive environments. In addition, customers greatly value the product's single-step application ability using conventional activators, eliminating the need to create an additional inventory of new activators.

Each year, Frost & Sullivan presents a Company of the Year award to the organization that demonstrates excellence in terms of growth strategy and implementation in its field. The award recognizes a high degree of innovation with products and technologies and the resulting leadership in customer value and market penetration.

Frost & Sullivan Best Practices awards recognize companies in various regional and global markets for demonstrating outstanding achievement and superior performance in leadership, technological innovation, customer service and strategic product development. Industry analysts compare market participants and measure performance through in-depth interviews, analyses and extensive secondary research to identify best practices in the industry.

Click here for more information about Axalta Mobility Coatings.

For more information about Frost & Sullivan, visit

To learn more about Frost & Sullivan Best Practices Recognition, click here.

About Axalta Coating Systems

Axalta is a global leader in the coatings industry, providing customers with innovative, colorful, beautiful and sustainable coatings solutions. From light vehicles, commercial vehicles and refinish applications to electric motors, building facades and other industrial applications, our coatings are designed to prevent corrosion, increase productivity and enhance durability. With more than 150 years of experience in the coatings industry, the global team at Axalta continues to find ways to serve our more than 100,000 customers in over 130 countries better every day with the finest coatings, application systems and technology. For more information visit and follow us @axalta on Twitter.

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NuFace Refocuses on Skin Care

NuFace is overhauling its product lineup, starting with skin care.

The brand, which has brought microcurrent facial technology to the masses, is rethinking its device-first strategy. The first prong of its efforts are two revamped conductive gels, now called the Firming + Brightening Silk Crème and the Hydrating Aqua Gel, which both double as leave-on treatments with skin care ingredients. Priced from $29 to $59 depending on size, the products are available at all of the brand’s retail and professional partners — including Sephora, Nordstrom, BlueMercury and Neiman Marcus.

The brand has confirmed projected retail sales for 2021 on track at the $150 million mark, with 20-25 percent of revenues coming from skincare. Executives didn’t comment on the figures, but noted the brand is bullish on the category. “Looking at our future in the current state of the business, this line is always driven by efficacy. Today, you have the device, but we envision a day as we launch into skin care to have a full lineup that makes up half of our revenue,” said Mike Larrain, chief executive officer of NuFace.

NuFace’s foray into skin care is centered around a proprietary You are unable to access this email address called IonPlex, which conducts the devices’ microcurrent into the skin for optimal results. “We are so strong in listening to clients and interacting, whether it’s online or in-store, and this is really just taking client feedback,” said Tera Peterson, cofounder and chief creative officer of NuFace. 

To that end, Peterson’s philosophy in developing the new gels — and its upcoming launches in the topicals — was also centered around feedback. “Microcurrent is more of a holistic approach to antiaging and is a very clean technology,” Peterson said. “So, we wanted to introduce clean, ionized skin care.”

To that end, NuFace is repositioning itself  as a “holistic beauty company,” which Larrain said will attract new swaths of consumers. “We’re going from being this device tech company to this holistic beauty company, and as we broaden out, we’ll be launching a line of boosters, which you can think of as a line of serums. In a couple years, an ionized cleanser or exfoliant, and then all the way into retinols for a full ionized line,” he said.

“The path to purchase for devices is typically longer than going out and buying a moisturizer, as you can imagine, based on the price points,” Larrain continued. “We do think there’s an opportunity to bring a great deal more consumers into the brand. We’ve sold over 3 million devices now. There’s an opportunity for us to educate the world on microcurrent since everyone is a candidate.”

NuFace isn’t just rethinking its skin care. Its devices will relaunch by year’s end with Bluetooth capabilities You are unable to access this email address personalized treatments. “It’s this concept of technology and personalization,” Larrain said. “How can we provide really good education and guidance in a very complicated space to get your best results. That’s the vision for the brand.”

For more from, see:

P&G Beauty on Meeting Generational Skin Care Needs

EXCLUSIVE: Hyram Yarbro to Launch Skin Care Range

EXCLUSIVE: Madison Beer and Vanessa Hudgens Discuss Releasing Their Own Skin Care Line

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Resolving Host? How to Fix This When Your Browser Is Stuck

Ok so for about a week my computer has been royally pissing me off in the worst way possible.  My laptop is a vertitable Porsche of a system.  32GB of RAM… Intel i7 Processor… 1TB SSD… it isn’t new but it’s a top of the line system and I take better care of my laptop than I do my own body (okay, that’s not entirely true… well maybe).  Today I met my match with a “Resolving Host” issue… let me explain.

The internet service we have at home is 1st Mass Mailer 2.3 crack serial keygen Everything is fast. I’m not one of those stupid click-happy users who downloads spyware, malware and potentially unwanted programs.  I know how malware works (heck, I even reverse engineer it in my spare time using IDA You are unable to access this email address and gdb).  I’m not a novice or new user.  I pay attention to what I click.

But here’s the thing: for the past month or so my computer has been slugging along and I never really took the time to figure out why.  And to be more exact: it wasn’t the computer itself but rather my browsers.

My main go to browser is Google Chrome.  I have Microsoft Edge and Firefox Quantum but I still use Chrome because I’m used to it and all my extensions live there.

Sometimes rebooting my computer would temporarily speed things up but it didn’t always fix things.

Now before I get into my issue and how I fixed it I need you to know something about me.  I used to work the Help Desk in college and I did Tier 2 and Tier 3 technical support at IBM as my first job.  I know how this stuff works.

But what really pisses me off is when I need to use Help Desk skills on my personal computer.  It’s like I have this innate believe that tech guys should be immune to computer problems.  It’s like I’m blaming God saying, “Hey, this can happen to everyone else but not me!  It’s not fair!” wha wha wha!  And then I start whining like a baby.

So whenever my computer starts acting weird I usually don’t apply the same critical thinking skills to my personal machine that I do to a client or customer.

Anyway, over the past few weeks my browser would continually say, “Resolving Host” in the status bar.

It was the most annoying thing in the world.  I pop open Google Chrome, and it says, You are unable to access this email address Host” in the status bar.  And it just hangs there… it doesn’t matter if I refresh the page or close and open the browser, Chrome is stuck in “Resolving Host“.

Chrome Stuck Resolving Host

After a few minutes it would sometimes fix itself but I couldn’t find any rhyme or reason to it.

Until today.

How to Fix “Resolving Host…” In Chrome

So here’s the thing – if you’ve ever You are unable to access this email address this error the first thing you need to fix is yourself: stay calm.  There is hope.

Resolving Host means there’s a problem… resolving the host.

To be more precise, it means there’s a problem with DNS.  The Domain Name Service, is the internet service that’s responsible for translating domain names, such as to an IP address.  If this step fails you’ll never be able to browse any where.


One other thing: when you see this “Resolving Host” issue sometimes the page does eventually error out.  In my case, it displayed the following error:

This site can't be reached's server IP address could not be found. Try: Checking the proxy, firewall, and DNS configuration Running Windows Network Disgnostics DNS_PROBE_FINISHED_BAD_CONFIG



And yes, I actually clicked the “Checking the proxy, firewall, and DNS configuration” and “Running Windows Network Diagnostics” links and neither helped one bit.

So here’s how I fixed this super annoying issue MRT Dongle 3.26 loader Archives here’s what you need to do right now to fix this problem.”

First we need to open an elevated command prompt.

Hit the Windows Key, type “cmd” and press Ctrl + Shift + Enter.

1. Flush DNS

This is a quick way to open the command prompt as an Administrator.  Now we’re going to flush the local DNS cache on your system.  If the local cache gets corrupted it can affect your ability to browse the web because the name-to-IP mappings will be invalid.

Bust open the command prompt and type this exactly:

ipconfig /flushdns

ipconfig flush dns

Alright, good now we need to use netsh You are unable to access this email address reset your IP settings.

Using netsh to reset TCP/IP

We’re going to type:

netsh int ip reset

yeah baby!

netsh int ip reset

netsh is a command line scripting utility that system admins use to automated tasks and configure various aspects of the local computer.  We’re using this magical tool to reset our TCP/IP protocol stack.  This trick is a lifesaver – it saved my but tonight – and that’s why I’m sharing it with you.

After it completes it’ll ask you to reboot.

You can type:

shutdown /r /t 0

That’s a zero not an “oh”.  It says, restart the computer and wait zero seconds.  In other words, do it now.

When the box comes back up all should be well.

What to do if that didn’t work

If you’re still having problems resolving host after following my above suggestions then make sure:

You aren’t using a Proxy.  Hit the Windows Key, type “inetcpl.cpl”, hit the “Connections” tab, choose “LAN Settings” and make sure everything is unchecked.  Everything, meaning: “Automatically detect settings”, “Use Automatic Configuration Script” and “Use a proxy server for your LAN”

Internet Properties Windows 10

Secondly, make sure you’re IP and DNS settings are set to use DHCP.

Hit the Windows Key again, type “ncpa.cpl”, double-click your Adapter, go to “Properties”, double click “Internet Protocol Version 4 (TCP/IPv4) and make sure everything is set to “Obtain an IP address automatically” and “Obtain DNS server address automatically”

Windows DHCP

If you still have the problem after making this change, either reboot your internet router or manually change your DNS server to, or

Sounds crazy I know but just do it.  Sorry to sound mean – but it works.

Alright that’s all I have I hope this was helpful – if it was remember to share this post with your friends and leave a comment!  Thanks!

Oh yeah, and Happy New Year! hehe.

Posted in Desktops, How To, Laptops, Web Browsers, Windows 10, Windows 7, Windows 8, Windows 8.1, Windows Vista Tagged with: Browsers

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