Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

BACKGROUND: Hypoxia-induced mitogenic factor (HIMF), a lung-specific growth factor, promotes vascular tubule formation in a matrigel plug model. We initially found that HIMF enhances vascular endothelial growth factor (VEGF) expression in lung epithelial cells. In present work, we tested whether HIMF modulates expression of fetal liver kinase-1 (Flk-1) in endothelial cells, and dissected the possible signaling pathways that link HIMF to Flk-1 upregulation. METHODS: Recombinant HIMF protein was intratracheally instilled into adult mouse lungs, Flk-1 expression was examined by immunohistochemistry and Western blot. The promoter-luciferase reporter assay and real-time RT-PCR were performed to examine the effects of HIMF on Flk-1 expression in mouse endothelial cell line SVEC 4–10. The activation of NF-kappa B (NF-κB) and phosphorylation of Akt, IKK, and IκBα were examined by luciferase assay and Western blot, respectively. RESULTS: Intratracheal instillation of HIMF protein resulted in a significant increase of Flk-1 production in lung tissues. Stimulation of SVEC 4–10 cells by HIMF resulted in increased phosphorylation of IKK and IκBα, leading to activation of NF-κB. Blocking NF-κB signaling pathway by dominant-negative mutants of IKK and IκBα suppressed HIMF-induced Flk-1 upregulation. Mutation or deletion of NF-κB binding site within Flk-1 promoter also abolished HIMF-induced Flk-1 expression in SVEC 4–10 cells. Furthermore, HIMF strongly induced phosphorylation of Akt. A dominant-negative mutant of PI-3K, Δp85, as well as PI-3K inhibitor LY294002, blocked HIMF-induced NF-κB activation and attenuated Flk-1 production. CONCLUSION: These results suggest that HIMF upregulates Flk-1 expression in endothelial cells in a PI-3K/Akt-NF-κB signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis

Pleiotropic effects of statins in distal human pulmonary artery smooth muscle cells

<p>Abstract</p> <p>Background</p> <p>Recent clinical data suggest statins have transient but significant effects in patients with pulmonary arterial hypertension. In this study we explored the molecular effects of statins on distal human pulmonary artery smooth muscle cells (PASMCs) and their relevance to proliferation and apoptosis in pulmonary arterial hypertension.</p> <p>Methods</p> <p>Primary distal human PASMCs from patients and controls were treated with lipophilic (simvastatin, atorvastatin, mevastatin and fluvastatin), lipophobic (pravastatin) and nitric-oxide releasing statins and studied in terms of their DNA synthesis, proliferation, apoptosis, matrix metalloproteinase-9 and endothelin-1 release.</p> <p>Results</p> <p>Treatment of human PASMCs with selected statins inhibited DNA synthesis, proliferation and matrix metalloproteinase-9 production in a concentration-dependent manner. Statins differed in their effectiveness, the rank order of anti-mitogenic potency being simvastatin > atorvastatin > > pravastatin. Nevertheless, a novel nitric oxide-releasing derivative of pravastatin (NCX 6550) was effective. Lipophilic statins, such as simvastatin, also enhanced the anti-proliferative effects of iloprost and sildenafil, promoted apoptosis and inhibited the release of the mitogen and survival factor endothelin-1. These effects were reversed by mevalonate and the isoprenoid intermediate geranylgeranylpyrophosphate and were mimicked by inhibitors of the Rho and Rho-kinase.</p> <p>Conclusions</p> <p>Lipophilic statins exert direct effects on distal human PASMCs and are likely to involve inhibition of Rho GTPase signalling. These findings compliment some of the recently documented effects in patients with pulmonary arterial hypertension.</p

Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension.

Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. Here, we identify BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH who bear mutations in the gene encoding BMPR-II, BMPR2. Mice bearing a heterozygous knock-in allele of a human BMPR2 mutation, R899X, which we generated as an animal model of PAH caused by BMPR-II deficiency, spontaneously developed PAH. Administration of BMP9 reversed established PAH in these mice, as well as in two other experimental PAH models, in which PAH develops in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a new therapeutic strategy for PAH

Distribution of macronutrients within organically grown Jerusalem artichoke (Helianthus tuberosus L.) tubers throughout the growing period

The aim of this research was to evaluate the distribution of macronutrients in organically grown Jerusalem artichoke (Helianthus tuberosus L.) tubers throughout the plant growing season. During the years of 2012 - 2014, the following Jerusalem artichoke cultivars were grown for our investigations on an organic farm in the south of Lithuania: Sauliai, Albik and Rubik. The soil had the following agrochemical characteristics: soil pHKCl 6.7-7.5, available phosphorus 101-232 mg kg-1, and available potassium 65-98 mg kg-1. Additional fertilizing was applied to maintain such agrochemical characteristics during all research years. Samples were taken eight times during the growing season of tubers: in March-June (spring period) and August-November (autumn period). Dry matter (DM) content was determined by drying samples to the constant weight at the temperature of 105°C; N – by Kjeldahl method; P – photometrically after wet digestion in sulphuric acid; K, Mg – by atomic absorption spectrmetry (using an Analyst 200). The data were statistically treated using ANOVA data analysis and the management module of the integrated system Statistica. For the evaluation of the analytical results, one-factor analysis of variance was carried out. Averages of separate treatments were calculated, the standard deviation and the least significant difference at a 95% probability level were estimated using the Fisher’s LSD test (P < 0.05). The distribution of macronutrients in organic Jerusalem artichoke tubers throughout the plant growing period is largely associated with their organogenesis stage. The highest dry matter and nitrogen content were established in tubers of the cultivars Sauliai and Albik in March, while the highest content of potassium and magnesium was determined in tubers of the cultivars Rubik and Sauliai in May. The highest phosphorus content was in the tubers of cv. Rubik in November

Distribution of macronutrients within organically grown Jerusalem artichoke (Helianthus tuberosus L.) tubers throughout the growing period

The aim of this research was to evaluate the distribution of macronutrients in organically grown Jerusalem artichoke (Helianthus tuberosus L.) tubers throughout the plant growing season. During the years of 2012 - 2014, the following Jerusalem artichoke cultivars were grown for our investigations on an organic farm in the south of Lithuania: Sauliai, Albik and Rubik. The soil had the following agrochemical characteristics: soil pHKCl 6.7-7.5, available phosphorus 101-232 mg kg-1, and available potassium 65-98 mg kg-1. Additional fertilizing was applied to maintain such agrochemical characteristics during all research years. Samples were taken eight times during the growing season of tubers: in March-June (spring period) and August-November (autumn period). Dry matter (DM) content was determined by drying samples to the constant weight at the temperature of 105°C; N – by Kjeldahl method; P – photometrically after wet digestion in sulphuric acid; K, Mg – by atomic absorption spectrmetry (using an Analyst 200). The data were statistically treated using ANOVA data analysis and the management module of the integrated system Statistica. For the evaluation of the analytical results, one-factor analysis of variance was carried out. Averages of separate treatments were calculated, the standard deviation and the least significant difference at a 95% probability level were estimated using the Fisher’s LSD test (P < 0.05). The distribution of macronutrients in organic Jerusalem artichoke tubers throughout the plant growing period is largely associated with their organogenesis stage. The highest dry matter and nitrogen content were established in tubers of the cultivars Sauliai and Albik in March, while the highest content of potassium and magnesium was determined in tubers of the cultivars Rubik and Sauliai in May. The highest phosphorus content was in the tubers of cv. Rubik in November