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The efficacy of Phosphodiesterase 5 (PDE5) inhibitors to re-establish endothelial function is reduced in diabetic patients. Recent evidences suggest that therapy with PDE5 inhibitors, i.e. sildenafil, may increase the expression of nitric oxide synthase (NOS) proteins in the heart and cardiomyocytes. In this study we analyzed the effect of sildenafil on endothelial cells in insulin resistance conditions in vitro.
Methodology/Principal Findings
Human umbilical vein endothelial cells (HUVECs) were treated with insulin in presence of glucose 30 mM (HG) and glucosamine 10 mM (Gluc-N) with or without sildenafil. Insulin increased the expression of PDE5 and eNOS mRNA assayed by Real time-PCR. Cytofluorimetric analysis showed that sildenafil significantly increased NO production in basal condition. This effect was partially inhibited by the PI3K inhibitor LY 294002 and completely inhibited by the NOS inhibitor L-NAME. Akt-1 and eNOS activation was reduced in conditions mimicking insulin resistance and completely restored by sildenafil treatment. Conversely sildenafil treatment can counteract this noxious effect by increasing NO production through eNOS activation and reducing oxidative stress induced by hyperglycaemia and glucosamine.
Citation: Mammi C, Pastore D, Lombardo MF, Ferrelli F, Caprio M, Consoli C, et al. (2011) Sildenafil Reduces Insulin-Resistance in Human Endothelial Cells. PLoS ONE 6(1): e14542. https://doi.org/10.1371/journal.pone.0014542
Editor: Maurizio C. Capogrossi, Istituto Dermopatico dell'Immacolata, Italy
Received: July 2, 2010; Accepted: December 16, 2010; Published: January 28, 2011
Copyright: © 2011 Mammi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work has been funded by Institutional funding from University Tor Vergata, PRIN 2007 (Progetti Ricerca Interesse Nazionale) Ministero dell'Università e della Ricerca, 2007, to A. F.) and by Department of Health RF 2007 (Tremoli), Fondazione Roma - Rome Foundation Research Grant 2008–2009. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Nitric oxide (NO) is a reactive free radical gas acting as an intracellular or extracellular messenger. It is synthesized from L-arginine by a family of isoforms of NO synthases (NOS 1–3). NO generation by NOS-3 (eNOS) in endothelial cells explains the effects of endothelial-dependent vasodilators on vascular relaxation and decreased platelet adhesion and aggregation. These and others effects of NO are mediated by increased cyclic GMP (c-GMP) formation due to soluble guanylyl cyclase activation.
In turn c-GMP activates protein kinase G (PKG) and through the phosphorylation of its substrates can regulate many other biological processes. The increase of c-GMP is counteracted by Phosphodiesterase 5 (PDE5) activity that transformes c-GMP into GMP.
e-NOS activity is regulated by agonist-induced elevation of intracellular free Ca2+ concentration with subsequent binding of Ca2+/Calmodulin (Ca2+/CaM) to e-NOS. However shear stress and isometric contraction have been shown to activate the enzyme in a Ca2+/CaM-independent manner [1], through the activation of c-GMP dependent PKG [2]. This mechanism may serve to ensure continuous production of NO, independently of fluctuations in intracellular Ca2+ levels. Although eNOS was initially reported to be phosphorylated exclusively on serine residues [3] other evidence has shown that eNOS is also phosphorylated in threonine- and tyrosine-domains [4]. NO production in endothelial cells is regulated by phosphorylation of several eNOS consensus sequence sites by means of protein kinase (PK) Akt-1, PKC and calmodulin kinase II [5].
In addition to its well known metabolic action, insulin has vasodilatory effects that depend on NO production in the vascular endothelium. It has been demonstrated that e-NOS activation induced by insulin is mediated by the PI3K-Akt pathway resulting in e-NOS phosphorylation in Ser1177 independently of Ca2+-CaM pathway [6]. Moreover insulin inhibition of PI3K/Akt-1 pathway leads to impaired NO availability [7], [8]. Reduced insulin-mediated vasodilatation might contribute to vascular damage in insulin-resistant states. We already demonstrated that hyperglycemia, via hexosamine pathway activation, induces selective insulin resistance in endothelial cells and impairs e-NOS activation [9].
Sildenafil citrate is a PDE5 inhibitor (PDE-5i) that belongs to the "new class" of PDE-5i. PDE-5 selectively degrades cyclic guanosine monophosphate (cGMP), hence its inhibition by sildenafil raises cGMP intracellular levels inducing vasodilatation. In recent years, several studies have shown that sildenafil initially used for the treatment of erectile dysfunction, may have other therapeutic applications [10]–[16]. Because of its potent vasodilatory action, sildenafil has also been extensively studied for treating primary or hypoxia-induced pulmonary hypertension [11], [12], [14], [15], [17].
Endothelial dysfunction is an important defect that contributes to erectile dysfunction and vascular disease in diabetes and is associated with insulin resistance [18]. It has been reported that sildenafil can improve endothelial dysfunction after acute and chronic treatment [19]. In diabetic rats PDE-5i (SK-3530) can activate Akt-1 signaling and inhibit proapoptotic stimuli maintaining erectile function [20].
A recent report showed that chronic treatment with sildenafil improve glucose metabolism in a model of high-fat fed mice, without directly affecting Akt-1 phosphorylation [21]. The molecular mechanisms for these data still await demonstration; a possible involvement of cGMP pathway in insulin signal transduction could be hypothesized.
Since several reports have shown that endothelial dysfunction is associated with insulin resistance [22]–[24], the aim of the present study was to investigate the role of sildenafil in NO production and changes in insulin signaling in HUVECs in basal and insulin resistance conditions.
Human umbilical endothelial cells (HUVECs), human arterial endothelial cells (HAEC) EBM-2 and EGM-2MV media were purchased from Lonza (Walkersville, MD, USA). Human fetal corpora cavernosa smooth muscle cells (hfCC) were a kind gift of Prof. Mario Maggi. Phospho-Akt-1 (Ser-473 and Thr-308), Akt-1, phospho eNOS (Ser-1177), eNOS and iNOS antibodies were obtained from Cell Signaling (Waltham, Mass). TRIZOL, M-MLV reverse transcriptase and Platinium High Fidelity Taq were purchased from Invitrogen (Paisley, UK). BSA, Arginine, Glucose and Glucosamine (Gluc-N) were obtained from Sigma (St Louis, Mo). LY 294002, nitro-L-arginine methyl ester (L-NAME) a non selective inhibitor of all NOS isoenzymes (26) and DAF-2DA were purchased from Calbiochem (San Diego, CA). Sildenafil powder was obtained from Viagra pills (Pfizer Inc); pills were ground into powder and dissolved in deionized H2O. The drug solution was filtered (0.45 µm pore size) and the solution was applied to a 285 mL Sephadex G-25 (superfine) column, equilibrated before using in deionized H2O at 20°C [25]. Sildenafil was eluted with 500 mL of 1% formic acid, lyophilized and resuspended in deionized H2O, and then was used to stimulate cell cultures. The Sildenafil concentration of 1µM was chosen based on data from Luedders et al. [26].
Cell culture
HUVEC and HAEC were growth in EGM-2MV at 37°C in an atmosphere of 95% air–5% CO2. HfCC were growth in M199 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and antibiotics (50 U of penicillin and 50 µg of streptomycin/ml). HUVEC and HAEC from passages 3 to 6 were cultured for 72 hours in presence of 5.5 mmol/L glucose, 30 mmol/L glucose (high glucose; HG), 10 mmol/L Gluc-N with or without 1 µM of sildenafil (5 or 72 h) and/or 10−7 M insulin. LY 2940002 (30 µM) was used to block the PI3K and inhibit the PI3K/Akt pathway.
RT-PCR analysis
The expression of endogenous PDE5 was determined by reverse transcription (RT) of total RNA followed by PCR analysis. Total RNA was extracted using TRIZOL. One microgram of total RNA was reverse transcribed using M-MLV reverse transcriptase according to the manufacturer's protocol. PCR of the cDNA was performed using Platinium High Fidelity Taq using the following pairs of primers:
- PDE5 sense: (5′-3′) ACC GCT ATT CCC TGT TCC TT
- PDE5 antisense: (5′-3′) AAG GTC AAG CAG CAC CTG AT
PCR parameters were: 35 cycles (94°C for 45s, 58°C for 45s, and 72°C for 1 min).
The PCR conditions were optimized for actin gene used as control.
Real-Time Quantitative PCR
Single-stranded cDNA was synthesized from 4µg total RNA samples using High-Capacity cDNA Archive Kit (Applied Biosystems). cDNA (40 ng) was amplified by RT-PCR using ABI PRISM 9700 System and TaqMan reagents. The 20× Assays-on-demand gene expression used were: PDE-5 (Hs00903251_m1), iNOS (Hs00167248_m1), and eNOS (Hs00167166_m1). Human RNA 18s were used for samples normalization. Each reaction was carried out in triplicate.
Western Blot Analysis
Cells were incubated for four hours in EBM-2 additioned with 0,1% BSA. After starvation cells were incubated in EGM-2MV with HG or with 10 mM Gluc-N to induce insulin resistance for 72 h with or without 1 µM sildenafil, added every 24 h. Cells were treated with 100 nM insulin during the last 30 min. of incubation. Extracts were obtained by lysing the cells in lysis buffer (50 mM Tris-HCl pH 7.6, 100 mM NaCl, 2 mM EDTA, 1 mM MgCl2, 1 mM CaCl2, 1% Triton X-100, 10% glycerol, 100 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 2 mM sodium ortovanadate, 5 mM sodium pyrophosphate and protease inhibitor). Protein concentration was determined by the method of Bradford, using bovine serum albumin (BSA) as a standard. 100 µg of proteins from each lysate were separated on a 10% SDS/PAGE gel and transferred to nitrocellulose membranes. Then membranes were incubated with the following antibodies: anti-Akt-1, anti-phospho Ser-473 Akt-1, anti-eNOS, anti-phospho Ser-1177 eNOS, anti-iNOS. The primary antibody was visualized using horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibodies and enhanced chemiluminescence.
NO production determination
The FACScalibur (Fluorescent Activated Cell Sorting, Becton Dickinson) was used to quantify NO production. Endothelial cells were washed with PBS and starved for 1 h at 37°C in M199 phenol free medium plus 0.1% BSA. After 30 min. different inhibitors were added when required (LY 294002 30 µM, L-NAME 1 µM). Then 30 min. later sildenafil (1 µM) was supplemented when demanded. Arginine (100 µM) was incubated 1h later and after 1h diaminofluoresceine-2 diacetate (DAF-2DA, 5 µM) probe was added. After 20 min. insulin (100 nM) has been added for 1h. Finally endothelial cells were collected and analyzed with FACS, by the Cellquest program (Becton Dickinson).
Statistical analysis of data
Data are representative of three or more independent experiments. Results are reported as mean ± SE or as percent increase respect to control where appropriate. Differences between treatments were analysed by ANOVA followed by Student's test and Mann Whitney test. A p value<0.05 was considered significant.
PDE5 expression in HUVECs
PDE-5 mRNA expression, measured in HUVECs cell line by RT-PCR, was detectable compared to the expression in human foetal corpora cavernosa (hfCC) cells used as positive control (Fig. 1A). Furthermore, Western Blot studies showed a significant expression of PDE-5 protein (Fig. 1B).
A: PDE5 mRNA expression in HUVEC and hfCC cells: RT-PCR shows that HUVEC express detectable levels of PDE-5 mRNA. Actin was used as control. B: Western Blot analysis for PDE5 protein expression. C: Real time PCR shows that insulin (30 min) and/or sildenafil (5 h) treatments induce PDE5 and eNOS but not iNOS expression *p<0.05 vs Ctrl, **p<0.01 vs Ctrl; † p<0.05 vs sildenafil. D: A representative Western Blot shows that insulin (30 min) and/or sildenafil (5 h) treatments do not alter iNOS protein levels.
Insulin up-regulates eNOS and PDE5, whereas sildenafil modulates only eNOS expression in HUVECs
Treatment with sildenafil (1 µM/5 h), insulin (100 nM/30 min), alone and in combination induced a 2-fold increase of eNOS but not iNOS mRNA expression, that remained roughly unchanged after treatment. iNOS protein expression was also unchanged in the same conditions (Fig. 1D). Interestingly, the positive effect of sildenafil on eNOS expression was not amplified by co-treatment with insulin. PDE-5 mRNA expression was up regulated (∼3 fold) by insulin alone or in combination with sildenafil (∼2.5 fold, Fig. 1C), whereas sildenafil alone did not significantly modify it. These data show that in our cellular model PDE5 expression is responsive to insulin treatment. Also, HUVEC cells respond to sildenafil in terms of eNOS expression in accordance with previous data, which showed that sildenafil increases eNOS expression in pulmonary vessels [27].
Sildenafil increases Akt-1 phosphorylation in insulin-resistance conditions
In order to validate our model of insulin resistance, we measured Akt-1 Ser473 phosphorylation in HUVECs cultured in HG and Gluc-N for 72 h. In keeping with previous observations, Akt-1 phosphorylation induced by insulin is impaired in HUVECs exposed to HG (30 mM) or Gluc-N (10 mM) [28], [29] respectively of ∼27% and ∼37% (Figure 2). Interestingly, in the same experimental conditions sildenafil co-treatment (1µM/72h) restored the ability of insulin to stimulate Akt-1 phosphorylation in presence of HG (+75% vs HG+insulin) or Gluc-N (+38% vs Gluc-N+insulin).
Insulin plus sildenafil treatment (1 µM/72 h) enhances Akt-1 phosphorylation in insulin resistance condition respect to the same conditions without sildenafil. Full bars represent conditions without sildenafil, dashed bars represent sildenafil treatment. Ctrl was expressed as 100%. Means ± S.E. (n = 5). ‡ p<0.05 vs insulin; †p<0.05 vs HG+insulin; § p<0.05 vs Gluc-N+insulin.
Sildenafil improves eNOS Ser1177 phosphorylation in basal and in insulin-resistance conditions
Since eNOS is a downstream effector of Akt-1, we studied eNOS Ser1177 phosphorylation in the same conditions shown in figure 2. Interestingly chronic (1 µM/72 h) and acute (1µ M/5 h) sildenafil treatment enhanced eNOS Ser1177 phosphorylation by ∼2 and 2.2 fold respectively (Fig. 3 and Fig. 4A) (p<0.05) under basal conditions. Moreover eNOS phosphorylation induced by acute sildenafil treatment is inhibited by LY294002 a PI3K inhibitor (Figure 4A). In accordance with previous data [30], HG reduced insulin induced eNOS Ser1177 phosphorylation by 25% (p<0.05). Sildenafil exposure (1 µM/72 h) markedly increased eNOS phosphorylation in HG conditions, both in the absence (+83% p<0.05) and in the presence (+74%, p<0.05) of insulin co-treatment (Figure 3), differently from what observed for Akt-1 Ser473 phosphorylation, where sildenafil was efficient only in the presence of insulin. As expected, eNOS phosphorylation was decreased by Gluc-N, in the presence of insulin (29%, p<0.05) and co-treatment with sildenafil was unable to improve eNOS phosphorylation, differently to what was observed for Akt-1 phosphorylation (Figure 3).
Sildenafil chronic treatment (1 µM/72 h) enhances eNOS phosphorylation in basal condition and after HG treatment, but not significantly after Gluc-N treatment. Full bars represent conditions without sildenafil, dashed bars represent sildenafil treatment. Ctrl was expressed as 100%. Means ± S.E. (n = 5). *p<0.05 vs Ctrl, ‡ p<0.05 vs insulin; † p<0.05 vs HG; †† p<0.05 vs HG + insulin.
A: Western Blot analysis of eNOS Ser1177 phosphorylation. Means ± S.E. (n = 4). * p<0.05 vs Ctrl; ° p<0.05 vs insulin; **p<0.01 vs sildenafil; °°p<0.05 vs sildenafil+insulin. B: Sildenafil treatment (1 µM/5 h) induces NO production that is partially inhibited from LY. Means ± S.E. (n = 6). *p<0.05 vs Ctrl; **p<0.01 vs Ctrl; ‡ p<0.05 vs insulin; ¶ p<0.05 vs LY; ¶¶ p<0.05 vs LY+insulin; # p<0.05 and ## p<0.01 vs sildenafil; ∥ p<0.05 vs sildenafil+insulin. C: L-NAME effects on NO production induced by sildenafil treatment. L-NAME reduces NO production induced by acute treatment with sildenafil. Means ± S.E. (n = 6). *p<0.05 vs Ctrl; # p<0.05 vs sildenafil. Full bar represent conditions without sildenafil, dashed bars represent sildenafil treatment.
Treatment with sildenafil induces NO production, not exclusively via PI3K pathway
In order to confirm that the increase in eNOS Ser1177 phosphorylation induced by sildenafil displayed biological effects in term of NO production, we studied intracellular generation of NO by flow-cytometric analysis. Insulin (100 nM) stimulation for 1h induced a significant increase of NO production (p<0.05). Sildenafil treatment (1 mM/5 h) markedly enhanced NO production in HUVECs compared to control (p<0.01); such effect was ∼1.5-fold higher than that obtained with insulin. Co-treatment with insulin blunted the effect of sildenafil, probably because of the insulin-induced increase in PDE-5 expression, previously described in fig. 1. In order to evaluate if sildenafil effects were mediated by PI3K pathway, the same experiments were performed in the presence of LY294002, a PI3K inhibitor. LY treatment reversed insulin effect in the absence of sildenafil (p<0.05), as expected [31]. In the absence of insulin LY markedly blunted sildenafil-induced NO production (p<0.01). However, LY did not completely reverse the effect of sildenafil since NO levels were not reduced to the baseline levels. These data suggest that in our model sildenafil effects are not mediated only through the PI3K pathway (Figure 4B).
In order to confirm whether NO generation induced by sildenafil was due to eNOS activation, NO production studies were repeated in the presence of L-NAME, a NOS inhibitor. As expected, L-NAME treatment completely reversed to baseline levels both insulin- (data not shown) and sildenafil-induced NO generation (1µM/5h). (Figure 4 C). These results were confirmed also in HAEC (data not shown).
NO production in insulin-resistance conditions in presence of sildenafil
As already established [7], [8], in accordance with eNOS phosphorylation studies (figure 3), conditions simulating insulin resistance (HG and Gluc-N) markedly inhibited NO production induced by insulin in HUVECs (p<0.05). Sildenafil treatment (1 µM/72 h) significantly increased NO generation, both in the presence of HG (p<0.05) and Gluc-N (p<0.05). Moreover insulin blunted sildenafil-induced increase in NO generation under insulin resistance conditions. Such effect was significant in HG conditions (p<0.05), but did not reach statistical significance with Gluc-N (Figure 5A). Similar data were obtained in HAEC (Figure 5B).
Sildenafil treatment induces NO production in insulin resistance condition (HG and Gluc-N) in HUVEC (panel A) and HAEC (panel B). Full bars represent conditions without sildenafil, dashed bars represent sildenafil treatment. Means ± S.E. (n = 6). *p<0.05 and **p<0.01 vs Ctrl; ‡ p<0.05 and ‡‡ p<0.01 vs insulin; † p<0.05 vs HG; $ p<0.05 vs sildenafil; # p<0.05 vs sildenafil+HG; ∞ p<0.05 vs Gluc-N.
The present study confirms that HUVECs express PDE5 mRNA as previously reported [32] and shows for the first time that its expression is regulated by insulin. HUVECs acute treatment (5 h) with sildenafil induced a sharp increase in NO production independently of insulin stimulation. Furthermore, sildenafil was able to reverse the suppression in insulin-induced NO production, caused by the PI3-K inhibitor LY294002. Pre-treatment with L-NAME, a NOS inhibitor, completely blocks the burst of NO production induced by sildenafil, claiming for a NOS- dependent effect. Chronic treatment with sildenafil (72 h) increased NO production both in basal conditions as well as in insulin resistance conditions.
It is well known that insulin promotes vasodilation and increases blood flow by modulating eNOS activity and expression through activation of the PI3-K/Akt-1 signalling [8], [33]. As expected [9] insulin stimulation induced NO production in HUVECs and inhibition of PI3K pathway by LY294002 reduced insulin-stimulated NO production. Our results show for the first time that in endothelial cells sildenafil enhances NO production by PDE5 inhibition and eNOS activation through a PI3K dependent pathway. We hypothesize that sildenafil, through specific inhibition of PDE-5, could increase intracellular concentration of free Ca2+ by c-GMP. Moreover it is possible that sildenafil increases intracellular Ca2+ levels in a cGMP independent manner by activating K+ channels with large conductance (BKCa) [26]. It is known that the association of Ca2+/calmodulin complex with eNOS increases eNOS Ser1177 phosphorylation and decreases Thr495 dephosphorylation [34]. This mechanism could amplify NO production in response to the intracellular Ca2+ elevation. Our results show that sildenafil does not enhance Akt-1 phosphorylation in basal conditions, although Akt-1 Ser473phosphorylation has been shown to increase after sildenafil stimulation in striatal [35] and corpora cavernosa smooth muscle (20). Sildenafil differently, increases Akt-1 Ser473 phosphorylation in HUVECs when associated with insulin co-treatment in presence of higher oxidative stress levels (HG and Gluc-N), improving insulin action. These data are in accordance with previous studies demonstrating that sildenafil decreases ROS and nitrotyrosine cell production [36], [37]. Interestingly, in basal conditions sildenafil increased NO production without modifying Akt-1 phosphorylation (Fig. 2 and 3). Such effect was only partially reversed by PI3K inhibitor LY, without reaching baseline levels. This suggests a partial involvement of PI3K in the observed effect, whereas we can exclude the involvement of Akt-1 phosphorylation. It is possible that PI3K activates eNOS by modulating other kinases like atypical protein kinases C, p70 S6 kinase and SGK-1. These effects are insulin-independent. These data are in accordance with a work of Ayala, who found that in mice with an high fat diet sildenafil improved insulin sensitivity measured with hyperinsulinemic-euglycemic clamp with no increased levels of Akt-1 phosphorylation [21].
Hyperglycaemic impairment of nitric oxide (NO) production by endothelial cells is implicated in the effect of diabetes to increase cardiovascular disease risk. As already reported [38], [39], we found that in insulin resistance conditions, insulin treatment does not induce NO production; chronic treatment with sildenafil improves NO production in basal conditions as well as in insulin resistance conditions in e-NOS-dependent manner. This effect was reduced when insulin resistance was induced by Gluc-N. In these conditions, the positive effect of sildenafil on eNOS phosphorylation was completely inhibited by Gluc-N. However eNOS mediated NO production was partly restored even in presence of Gluc-N. These results could be explained by the different mechanism of glucotoxicity. In fact, it is known that Gluc-N, by activating hexosamine pathway, causes O-GlcNac modifications at protein sites which are involved in insulin signalling [40]. Interestingly, under insulin resistance conditions we observed that insulin blunted the increase in NO generation induced by sildenafil. We demonstrated that insulin treatment was able to up-regulate PDE5 mRNA expression in HUVEC. It is possible that insulin increased PDE-5 mRNA expression through the transcription factors Sp1, AP-1 and AP-2. Indeed, AP-1 consensus sequence has been located at – 485 from the starting PDE-5 gene expression sequence (http://variome.kobic.re.kr/SNPatPromoter/snpatpromoter.jsp?id=NM_001083).
As a consequence, it is possible that insulin might reduce intracellular cGMP through an increased activity of PDE5, with a consequent decrease in Ca2+/CaM dependent eNOS activation and subsequent NO production.
These findings indicate that sildenafil improves insulin signalling and NO production in endothelial cells, probably by reducing oxidative stress induced by hyperglycemia and by increasing intracellular Ca2+ levels.
The novel finding that insulin stimulates PDE-5 expression can explain the reduced response to sildenafil therapy in diabetic patients with elevated insulin resistance and hyperinsulinemia. Our data clarify the mechanism through which sildenafil improves insulin action in endothelial cells. Further investigation are necessary to discover all the substrates involved in this process and to understand if this novel action of sildenafil is specific for this molecule or it is a class effect of PDE-5i.
In conclusion, we hypothesize that sildenafil can be potentially used to improve insulin action in type 2 diabetic patients leading to new therapeutic strategies for type 2 diabetes and other related cardiovascular disease.
In Figure 6 we show a schematic summary of sildenafil action in endothelium.
Author Contributions
Conceived and designed the experiments: CM DL. Performed the experiments: CM DP ML FF CC LG DL. Analyzed the data: CM DP ML FF MC M. Fini M. Federici PS GD AB GMR AF DL. Contributed reagents/materials/analysis tools: M. Fini DL. Wrote the paper: CM DP MC CC LG DL. Performed several nitric oxide measurements and cell biology experiments: MT.
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- 32. Favot L, Keravis T, Holl V, Le Bec A, Lugnier C (2003) VEGF-induced HUVEC migration and proliferation are decreased by PDE2 and PDE4 inhibitors. Thromb Haemost 90: 334–343.
- 33. Kuboki K, Jiang ZY, Takahara N, Ha SW, Igarashi M, et al. (2000) Regulation of endothelial constitutive nitric oxide synthase gene expression in endothelial cells and in vivo : a specific vascular action of insulin. Circulation 101: 676–681.
- 34. Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R (2001) Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res 88: E68–E75.
- 35. Puerta E, Hervias I, Goni-Allo B, Lasheras B, Jordan J, et al. (2009) Phosphodiesterase 5 inhibitors prevent 3,4-methylenedioxymethamphetamine-induced 5-HT deficits in the rat. J Neurochem 108: 755–766.
- 36. Bivalacqua TJ, Sussan TE, Gebska MA, Strong TD, Berkowitz DE, et al. (2009) Sildenafil inhibits superoxide formation and prevents endothelial dysfunction in a mouse model of secondhand smoke induced erectile dysfunction. J Urol 181: 899–906.
- 37. Milara J, Juan G, Ortiz JL, Guijarro R, Losada M, et al. (2010) Cigarette smoke-induced pulmonary endothelial dysfunction is partially suppressed by sildenafil. Eur J Pharm Sci 39: 363–372.
- 38. Alvarado-Vasquez N, Zapata E, Alcazar-Leyva S, Masso F, Montano LF (2007) Reduced NO synthesis and eNOS mRNA expression in endothelial cells from newborns with a strong family history of type 2 diabetes. Diabetes Metab Res Rev.
- 39. Salt IP, Morrow VA, Brandie FM, Connell JM, Petrie JR (2003) High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser1177 phosphorylation in human aortic endothelial cells. J Biol Chem 278: 18791–18797.
- 40. Ball LE, Berkaw MN, Buse MG (2006) Identification of the major site of O-linked beta-N-acetylglucosamine modification in the C terminus of insulin receptor substrate-1. Mol Cell Proteomics 5: 313–323.


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Here are a dozen piece of information for the 2012 traveler.
- The Department of Transportation this month denied requests to delay implementation of this rule, so as of last week, you will know what you'll pay if you decide to check a bag or another piece of luggage.
- You won't immediately know how much your entire airline ticket will charge when you book online - at least not yet.
- You'll almost certainly be paying more for airline tickets. That's not a big shock; although oil prices teased us last fall by reducing to $80 a barrel, they're hovering around $100.
- Credit card companies will continue to court your business with tremendous flier-mile offers. 2011 may be remembered as the year British Airways seduced with the assure of 100,000 flier miles for signing up for a Visa card.
- You'll pay more for hotels in a few places, and Southern California will be one of them. In Los Angeles County, you'll fork over about $5 a night more for a room, up to an average of $132, and in San Francisco, the regular rate will rise by about $10 to $136.

subset: commoncrawl


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Spotsylvania in Spotsylvania County, Virginia — The American South (Mid-Atlantic)
Spotsylvania Court House
In the Path of War
— Lee vs. Grant – The 1864 Campaign —
The Civil War shattered Spotsylvania's rural tranquility. In August 1862, Union troops raided the village, and nine months later Confederate authorities converted the courthouse yard into a temporary stockade to hold Union prisoners captured at the Battle of Chancellorsville. Worse was yet to come.
In May 1864, the Union and Confederate armies collided just outside the village. Spotsylvania was under fire for two weeks before the armies moved on. In their wake, they left battered buildings, miles of earthworks, and more than 30,000 casualties.
"The place looked quite desolate, very few persons remained and what few houses remained standing were pretty well riddled with shot and shell."
- Union soldier, 1865.
Erected by Virginia Civil War Trails
Marker series. This marker is included in the Virginia Civil War Trails marker series.
Location. 38° 12.102′ N, 77° 35.337′ W. Marker is in Spotsylvania, Virginia, in Spotsylvania County. Marker is on Courthouse Road (Virginia Route 208) near Brock Road, on the right when traveling south. Touch for map. Marker is in this post office area: Spotsylvania VA 22553, United States of America. Touch for directions.
Other nearby markers. At least 8 other markers are within walking distance of this marker. A different marker also named Spotsylvania Court House (here, next to this marker); Spotsylvania County Honor Roll (within shouting distance of this marker); Battle of Spotsylvania (within shouting distance of this marker); Lee's Headquarters (within shouting distance of this marker); Spotsylvania Courthouse and Jail (about 300 feet away, measured in a direct line); Spotsylvania County Jail (about 400 feet away); Booth Hall (about 700 feet away); Confederate Soldiers (approx. 0.3 miles away). Touch for a list and map of all markers in Spotsylvania.
More about this marker. In the upper right George L. Frankenstein, a native German who served in the Union army, painted this watercolor of Spotsylvania Court House shortly after the war. Below the painting and in the center is a map of the area around Spotsylvania Court House with related sites indicated.
Categories. • Notable Events • War, US Civil •
More. Search the internet for Spotsylvania Court House.
Credits. This page was last revised on June 16, 2016. This page originally submitted on November 20, 2007, by Kevin W. of Stafford, Virginia. This page has been viewed 1,211 times since then and 8 times this year. Photos: 1. submitted on November 20, 2007, by Kevin W. of Stafford, Virginia. 2. submitted on January 11, 2009, by Bill Coughlin of North Arlington, New Jersey. 3. submitted on August 13, 2008, by Craig Swain of Leesburg, Virginia. 4. submitted on November 20, 2007, by Kevin W. of Stafford, Virginia. 5. submitted on August 13, 2008, by Craig Swain of Leesburg, Virginia.


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2019-2020 Tuition
Rates are per student. Discounts are given to students taking more than one class. Discount is per student, not per family.
1 class per week: $46 per month
Tuition: Payment is by the month and the amount will always remain the same no matter how many lessons you receive per month. Payment is due on the FIRST lesson of each month. If payment is not received by the start of the third class, the student will not be permitted into class and a 5% LATE FEE will be charged. If you have a problem with this arrangement please contact Stacey to arrange an alternate plan. YOU MUST PAY FOR MISSED LESSONS! Missed lessons may be made up in another class if you desire. Since we follow the school calendar, we take a Winter Recess, and Spring Break. Nevertheless, payments will remain the same. Discounts are given to students taking more than one class. Discounts are per child not per family. This year a registration fee of $20.00 per family will be charged to have each student placed on file. All boys receive one free trial month of dance. In an attempt to keep prices down, we accept cash and checks only.Scholarships: A scholarship fund has been set up for a limited number of students who truly want and love to dance, but can't afford classes. If you are in need of a dance scholarship, please contact Stacey for more information. Each year a Silver Star Scholarship, Jessica Taylor Memorial Scholarship, and a Nancy Amberg Memorial Scholarship will be awarded to three deserving students.
Attendance: Attendance will be taken in each class. Good attendance is essential for learning. Also, as mentioned above, missed lessons are still paid for. If you miss a class and would like to make it up, please contact Stacey for makeup times. Stacey reserves the right to refuse a student's privilege to perform based on attendance.


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Ben Meadows:
- Price:
- $102.75
- Description
- Specifications
- Reviews
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This fast-acting contact herbicide controls selected submersed, emergent, and floating aquatic weeds. Propeller is most effective when applied to young, actively growing weeds in water with a pH of less than 8.5. May be applied to quiescent or slow-moving bodies of water such as: bayous, canals, drainage ditches, lakes, marshes, ponds (including golf course ponds), and reservoirs. Breaks down quickly in water and leaves no accumulation in the sediment. Active Ingredient: 51% Flumioxazin.
- Restricted Sales: R93 Cannot be sold Internationally
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Two teens were arrested for an alleged armed robbery following a lengthy investigation by Norwalk Police.
Gabriel Banegas, 19, of Stamford, and Jeremy Jimenez, 19 of Norwalk, were apprehended by Norwalk detectives on Thursday, March 21, in connection with the March 11, armed robbery of a juvenile in the Colonial Villiage Housing Complex, said Norwalk Terrance Blake.
After a lengthy investigation, the Detective Bureau was able to secure arrest warrants for the two, Blake said.
Both were charged with robbery, conspiracy to commit robbery, and risk of injury to a minor. They are being held on $750,000 bond.
Detectives said they wanted to thank the community for their help and ask anyone with information regarding this investigation to contact Detective Brendan Collins at 203-854- 3191. Anonymous tips can be sent to the below contacts.
Anonymous Internet tips can be sent to the Norwalk Police website at www.norwalkpd.com .
Click here to sign up for Daily Voice's free daily emails and news alerts.


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"That is why a man leaves his father and mother and is united to his wife, and they become one flesh." Genesis 2:24
Dear Single Mamas:
Are you courting with the intention of marrying? Are you engaged? How exciting! It is beautiful to meet someone who loves you and your child. This kind of love is beautiful because your boyfriend or fiancé made a choice to pursue and love you and your child as you walk towards a Godly marriage.
But in pursuing a Godly marriage, it is important to understand and trust God's standard now. In the verse quoted above, God tells us that when you marry someone you become "one flesh" with them. Paul expands on oneness in the marriage relationship in Ephesians 5:21- 33. Specifically, Paul notes the way a Godly marriage emulates Christ's relationship with the Church. Christ's relationship with the Church was a covenant. Thus, we implicitly see that our marriage is meant to be a covenant.
In Matthew 19:5-6, Jesus himself quoted Genesis 2:24 and then said, "So they are no longer two, but one flesh. Therefore what God has joined together, let no one separate." Thus, you and your spouse will be one.
This standard of oneness applies equally to the single mother and the childless woman – when you marry him, you and he enter a covenant and become one flesh. Having a Godly marriage means preferring one another above all else, even though you had your child before you met your (potential) one-day husband. This can be tough, and it runs counter to popular culture. But, if we trust in God, we trust that His design for marriage.
A Godly marriage between you and your fiancé have the opportunity to express the true definition of "love." The word "love" as used throughout the Bible is defined as "agape," which is a deliberate choice to express God's nature of love, even when someone does not deserve it. As Strong's defines it, expressing agape love is not based on feelings or impulses (which shift depending on the circumstances), and may mean going against the natural inclination to choose a certain person over someone else.
Expressing agape love to your one-day husband can be tough as a single mom. I would know, as it took some time to re-program my mind and switch from what popular culture tells us – choose your child before everyone, even your spouse. But, we must make a conscious choice to go against our natural inclination to prefer our child over our spouse. Covenant is between a man and a woman, not a mother and her child.
By the time you read this article, I will be married to my fiancé. Even though I was a mother first for four, almost five years, I will be striving to pursue covenant and live as one flesh with my fiancé. It may be tough, but I trust in God's design for marriage. Will you trust God in your future marriage?
From special education classes to single motherhood to graduate of Emory University School of Law, Melonie Wright is proof that God is a Redeemer. When Wright was in preschool, her parents were told she was a slow learner and was placed in special education classes, citing she never talked in class and didn't respond as others her age did. Melonie encountered God after experiencing much heartache and adversity. As a former college athlete, abuse survivor, and single mom who once had only cents to her name, Melonie knows what it is to be an overcomer through Christ and has a passion to teach other single moms to do the same thing. Melonie lives in Birmingham, AL with her daughter, Raegan, where she practices commercial litigation. To read more about Melonie click here.
The Life of a Single Mom is a national, faith-based, nonprofit that exists to see that no single mom walks alone. To date, we have worked with more than 1,500 churches & community groups to start or improve a single mom's support group in cities throughout the U.S. and beyond. We have a large array of books, curriculum, training materials, and online instructional videos to support ministry leaders who lead single moms. Our single mom programs focus on empowering single moms to grow spiritually, emotionally, financially, and parentally through a number of projects including: Single Mom University, Single Moms Across America, the National TLSM Single Moms Conference, and a variety of programs throughout the U.S. For more information, visit www.thelifeofasinglemom.com


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Evidently men have no need to be aware of breast cancer: according to Cancer Research UK, they make up less than 1% of new cases in the UK [1], perhaps a sufficiently small minority to be largely ignored in terms of risk of developing breast cancer. Yet the same data set reveals that in 2007 there were more than 45,000 new cases in women, a substantial proportion of whom, it is safe to assume, will have fathers, husbands and sons who are significantly affected by breast cancer.
The divisive and unnecessary gender bias of this 'make cancer fun on Facebook' campaign isn't the only reason to take exception. The very notion of raising awareness of breast cancer in some directionless do-good sense renders this a pointless exercise. Highlighting the risks and the efficacy of self-examination would be an excellent idea; pointing out the need for greater investment in valid cancer research is a suitably worthy cause. However, it is arguable that this campaign can even succeed in challenging the terrible taboo that leaves many breast cancer patients socially isolated. Breasts, femininity and sexual attractiveness are so intricately and complexly tangled; add to this the still widely held belief that cancer kills and we are dealing with a monster that threatens our every instinct.
Or rather, we are not dealing with it at all. Soldiering on at the front line of 'the enemy that must not not be named' are organisations such as Macmillan Cancer Support [2]. They are brave and heroic and we admire them, although until we are inadvertently thrown into the line of fire, we do so from a very safe distance. We can dismiss the advances of most major killer diseases through some simple blame-laying - lifestyle aetiology can more readily be applied to HIV and heart disease than to the generally indiscriminate nature of cancer - a certain penchant for risk behaviours can sometimes do the trick here too.
Even with highly specific aims, cancer awareness campaigns are, at best, troublesome. Advertising undertaken by Macmillan and Cancer Research UK undoubtedly increases the profile of cancer and much-needed donations. "For just £4 a month...", the mantra of daytime TV, enables most of us to give regularly to charity without noticing the cost or having to think too much about what it is we are funding. Indeed, direct debit has become the most popular way of donating to charities [3], accounting for most of the £9.9 billion donated in 2009 in the UK alone, with the top causes being medical research and hospitals and hospices [4]. In spite of only half of the UK adult population donating and an 11.4% decrease in real terms from 2008, that's still an awful lot of money.
But there is something very wrong with this picture, for it is not us, the average Joes, who should be worrying about whether we can afford a couple of quid a month in order to morally avoid facing cancer. Consider this: if all 5.7 billion adults [5,6] on the earth gave £4 a month for a year (a ridiculous proposition), the total would still fall £56 billion short of the £256 billion annual revenue earned by the top 20 pharmaceutical companies in 2009 [7]. And it gets worse: whilst no. 1 company Pfizer's CEO Jeffrey Kindler warned shareholders against the dangers of using their dollars to line a single basket of golden eggs (or should that be purple diamonds?), the cut-throat world of pharma R&D, at a time when revenue is down (i.e. profit growth is less than in the previous year), means incomplete trials and a reluctance to invest in anything but 'dead cert' blockbusters [8].
A pertinent example here is the development of PLX4032, a trial drug for the treatment of advanced melanoma. In the early stages, melanoma treatment is relatively easy and successful. However, the one year prognosis and a history of failed trials for treatment of advanced melanoma have been met with a reluctance on the part of large pharmaceutical companies to invest in further research, according to Peter Hirth, CEO of Plexxikon - the small biotechnology company in California who developed PLX4032 [9]. It comes as no surprise then, that after early successes with small-scale trials of PLX4032, Plexxikon attracted the interests of larger companies and in 2006 reached an agreement with Swiss company Roche, "one of the leading pharmaceutical companies in oncology" [10], with an annual revenue of around $5.1 billion [11], placing them just outside the top 20, no doubt hoping to improve their chart position with a new blockbuster and most of the heavy legwork out of the way.
So, taken at face value, the innocent plea in my Facebook inbox is benign enough, but there is no ignoring the fact that in the midst of juggling our miniscule monthly incomes in the hope we can scrape off a little surplus and somehow beat cancer, we forget about the big, fat, rich benefactors of the pharmaceutical industry, who don't feel the guilt we do and are no more compelled to share intimate details about their underwear than they are to risk shareholders' money on treatments that don't have that Wow! factor. A vague call to raise awareness may lead to a few more charitable donations, which in essence is our consent to maintaining the status quo. Cancer is not a battle that can be fought on two fields and we foot soldiers must now become conscientious objectors.
And for the record, it's black and I like it behind me; both rather apt responses, all things considered.
* CIA (2010) statistics for aged 15 and over equate to approximately 73% of 6.9 billion global population (US Census Bureau, 2010).
- Cancer Research UK (2007) Breast Cancer - UK Incidence Statistics http://info.cancerresearchuk.org/cancerstats/types/breast/incidence/.
- Macmillan Cancer Support (2010) About Us. http://www.macmillan.org.uk/Aboutus/AboutUsHome.aspx.
- Goodey, L. and Harrison, R. (Charities Aid Foundation) (2006) Individual giving in the UK: findings and context. NCVO Conference Paper.
- 4. Charities Aid Foundation and National Council for Voluntary Organisations (2009) UK Giving 2009: An overview of charitiable giving in the UK, 2008/9.
- Central Intelligence Agency (2010) The World Factbook. https://www.cia.gov/library/publications/the-world-factbook/geos/xx.html#People.
- US Census Bureau (2010) World POPClock Projection. http://www.census.gov/ipc/www/popclockworld.html.
- Roth, G.Y. (2010). 2010 Top 20 Pharmaceutical Companies Report. http://www.contractpharma.com/articles/2010/07/2010-top-20-pharmaceutical-companies-report.
- Contract Pharma (2010) #1 Pfizer. http://www.contractpharma.com/articles/2010/07/pfizer
- Ledford, Heidi (2010) Rare victory in fight against melanoma. http://www.nature.com/news/2010/100907/full/467140b.html.
- Plexxikon (2006) Plexxikon and Roche enter partnership to develop targeted cancer therapeutic medicine PLX4032. http://www.plexxikon.com/view.cfm/58/Press-Releaseshttp://www.plexxikon.com/view.cfm/58/Press-Releases.
- Wikipedia (2010) Hoffman-La Roche. http://en.wikipedia.org/wiki/Hoffmann%E2%80%93La_Roche.


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Tag Archives: Coffee
The Rainy Kid's Show
This is a quick design for a poster I did for "The Rainy Kid Show" a benefit concert some friends and I put on at a local coffee shop in Colorado Springs called Jives. The show couldn't have gone better: … Continue reading
Posted in Art, Creativity, Design Tagged Children's Hope Chest, Coffee, Colorado Springs, Jives, Jives Coffee, Jives Coffee Shop, Rainy Kid Show, Rainy Kid's Show, Rising Voice Leave a comment
This Is Mugwalls!
A quick parody on a famous scene from the movie 300. We went to a local coffeeshop named Mugwalls and had a blastAn ARTISTS ProductionPersian-Osa OkLeonidas-Andrew KaiserDirector-Christopher Kaspar more about "This Is Mugwalls!", posted with vodpod


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Amsterdam photo of the day: proof that Simon Pegg is a time-traveler
Alternate title: Simon Pegg likes it ruff.
I did a double-take when I saw this portrait at the Museum Willet-Holthuysen (basically a rich 19th-century family's house that's been opened to the public). Because, look:
I mean, come on. Clearly Pegg has been for at least one jaunt in the TARDIS.
Or else… Oh my god… Like Keanu Reeves… Simon Pegg is immortal!
(I was so startled that I forgot to get any info about the painting itself, such as whom it purports to be a portrait of. And I can't find any info about it online. Drat.)


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