text:
Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) in chronic hypoxia- and antigen-mediated pulmonary vascular remodeling
© Angelini et al.; licensee BioMed Central Ltd. 2013
Received: 24 April 2012
Accepted: 12 December 2012
Published: 4 January 2013
Both chronic hypoxia and allergic inflammation induce vascular remodeling in the lung, but only chronic hypoxia appears to cause PH. We investigate the nature of the vascular remodeling and the expression and role of hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELMα) in explaining this differential response.
We induced pulmonary vascular remodeling through either chronic hypoxia or antigen sensitization and challenge. Mice were evaluated for markers of PH and pulmonary vascular remodeling throughout the lung vascular bed as well as HIMF expression and genomic analysis of whole lung.
Chronic hypoxia increased both mean pulmonary artery pressure (mPAP) and right ventricular (RV) hypertrophy; these changes were associated with increased muscularization and thickening of small pulmonary vessels throughout the lung vascular bed. Allergic inflammation, by contrast, had minimal effect on mPAP and produced no RV hypertrophy. Only peribronchial vessels were significantly thickened, and vessels within the lung periphery did not become muscularized. Genomic analysis revealed that HIMF was the most consistently upregulated gene in the lungs following both chronic hypoxia and antigen challenge. HIMF was upregulated in the airway epithelial and inflammatory cells in both models, but only chronic hypoxia induced HIMF upregulation in vascular tissue.
The results show that pulmonary vascular remodeling in mice induced by chronic hypoxia or antigen challenge is associated with marked increases in HIMF expression. The lack of HIMF expression in the vasculature of the lung and no vascular remodeling in the peripheral resistance vessels of the lung is likely to account for the failure to develop PH in the allergic inflammation model.
KeywordsPulmonary hypertension Hypoxia-induced mitogenic factor (HIMF) Chronic hypoxia Th2-mediated inflammation Vascular remodeling
Pulmonary hypertension (PH) is clinically defined by a mean pulmonary artery pressure (mPAP) ≥ 25 mmHg at rest [1, 2]. This condition can manifest itself as a primary disease (idiopathic with or without a genetic linkage) or as a complication of another disease such as collagen vascular disease (e.g., scleroderma), human immunodeficiency virus (HIV) infection, Shistosoma mansoni (S. mansoni) infection, or chronic obstructive pulmonary disease [1–4]. The exact initiating event in the pathogenesis of PH is largely unknown, but several conditions, including chronic hypoxia and chronic pulmonary inflammation, have been associated with the development of PH in humans [5–7]. These conditions induce endothelial cell dysfunction as well as endothelial and vascular smooth muscle cell proliferation and hypertrophy within the pulmonary circulation [8]; changes in the cells of the vasculature thicken pulmonary arteries and arterioles, narrowing their lumens, resulting in increases in pulmonary vascular pressure and resistance [5, 8, 9].
A growing body of evidence suggests that inflammation plays a role in the vascular remodeling associated with chronic hypoxia and other experimental manifestations of PH [10–14]; these findings have accelerated investigation into the potential mechanisms of inflammation in this disease process [7]. Burke et al.[11] reported the upregulation of several pro-inflammatory genes in the lungs, including stromal cell-derived factor-1 (SDF-1/CXCL12), monocyte chemoattractant protein-1 (MCP-1), and interleukin (IL)-6, as well as the accumulation of monocytes within the pulmonary vasculature during chronic hypoxia. Also, many studies have shown that T-cell helper 2 (Th2) cytokines, particularly IL-4 and IL-13, are involved in the pathogenesis of pulmonary vascular remodeling [13–18]. We have recently reported that IL-4 acts as a proangiogenic molecule in the lungs of mice during chronic hypoxia [13]. To examine the role of Th2 cytokines in pulmonary vascular remodeling, Daley et al.[15] modified the standard antigen-challenge model normally used to examine inflammatory asthma. In this modified model, extended antigen challenges with either ovalbumin (Ova) or Aspergillus fumigatus antigen (Asp ag) lacking any viable fungus produced severe pulmonary vascular remodeling involving the proliferation of vascular smooth muscle cells. This remodeling was reduced in both IL-4 knockout mice and mice that had IL-13 signaling neutralized [15]. Surprisingly, this model produced no increases in right ventricular systolic pressure. In an S. mansoni infection model of PH, Graham et al.[19] demonstrated that mice that lacked the IL-13 decoy receptor, IL-13Rα2, displayed more severe pulmonary vascular remodeling than did wild-type controls. They also reported an increase in right ventricle (RV) maximum pressure in the infected IL13Rα2 knockout mouse compared to uninfected wild-type controls. In a separate study, Swain et al.[17] reported pulmonary vascular remodeling and development of PH as a result of Pneumocystis pneumonia in both wild-type and CD4+ T-cell-depleted mice; notably, these pathological changes still occurred in IL-4 knockout mice, and IL-13 was not detected in the lungs of the mice during the persistent phase of the model [17]. These studies suggest a role for inflammation in pulmonary vascular remodeling, but currently, the exact involvement in this process is unclear.
Hypoxia-induced mitogenic factor (HIMF), also known as "found in inflammatory zone 1" (FIZZ1) or "resistin-like molecule alpha" (RELMα), is highly upregulated in the lung in response to both chronic hypoxia [10, 20, 21] and Th2-mediated inflammation [16, 17, 22–27]. We have demonstrated that HIMF has proliferative, angiogenic, vasoconstrictive, and chemokine-like properties that are associated with the development of PH [10, 20, 21, 28, 29]. We have also demonstrated that overexpression of HIMF within the lungs induces a pattern of vascular remodeling and hemodynamic changes similar to that in chronic hypoxia-induced PH and that the in vivo blockade of HIMF expression within the lung reduces the pathologic vascular and hemodynamic changes associated with this model [10, 20]. These data indicate that HIMF plays a direct role in the induction of pulmonary vascular remodeling and the development of PH associated with chronic hypoxia. HIMF is also upregulated in response to pulmonary inflammation [16, 17, 22–25, 27, 30]. It has been reported that HIMF expression is increased in the lungs of several models of Th2-dependent inflammation, including allergic asthma [15, 22, 23, 27], human herpes virus 8 infection [25], Pneumocystis pneumonia [17], S. mansoni infection [16, 19], and bleomycin-induced pulmonary fibrosis [24, 30]; all of these models are associated with pulmonary vascular remodeling. Our laboratory has demonstrated that a tail vein injection of recombinant murine HIMF into mice induces a pro-inflammatory state within the lungs associated with vascular remodeling [14] and that HIMF can induce de novo production of both SDF-1 and MCP-1 in cultured endothelial cells and ex vivo lung organ culture [14, 29]. We have also shown that the human isoform of HIMF, RELMβ, is upregulated in the lungs of patients diagnosed with scleroderma-associated PH [31]. In lung samples from these patients, RELMβ was expressed in inflammatory cells (macrophages, T-cells) as well as in myofibroblasts, endothelium, and vascular smooth muscle [31]. Renigunta et al.[32] also demonstrated that hypoxia could induce human RELMβ expression in both a lung cancer cell line (A549) and primary pulmonary artery smooth muscle cells in vitro.
At the current time, it remains unclear whether Th2 models of vascular remodeling induction, including Ova, Asp ag, and S. mansoni infection, actually cause the development of PH as chronic hypoxia does (e.g. increased mPAP, RV hypertrophy, vascular remodeling). In the current study, we directly compare chronic hypoxia- and Th2 inflammation-induced pulmonary vascular remodeling to address this issue and identify possible explanations of the observed differences.
Experimental animals
Adult male C57BL/6 mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) were used for all of the studies. The animal housing and experimental protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University. The mice had free access to food and water and were housed in a room with a 12:12 h light–dark cycle at 20–24°C.
Ova model of Th2-induced pulmonary vascular remodeling
The Ova used for this study was prepared as follows: Ova (Grade V; Sigma-Aldrich, St. Louis, MO) was diluted to 1 mg/ml in 0.15 M sterile saline that was complexed with Alum (Imject Alum; Thermo Fisher Scientific, Waltham, MA). Mice were injected i.p. with 50 μg Ova and 2 mg Alum or equivalent volume of sterile saline solution on day 0 of the experiment. This procedure was then repeated on day 14. On days 28–30, the mice were intranasally challenged with 50 μg Ova diluted in 50 μl of saline or with 50 μl saline alone. The intranasal challenges were also repeated on days 35–37, 41–43, and 45. Mice were sacrificed on day 46 of the experiment by isoflurane overdose, and tissue was processed as we have described [10, 20]. Mice used for hemodynamic measurement were anesthetized with an i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and mPAP was determined as described [20, 21]. All mice were euthanized by exsanguination. Bronchoalveolar lavage fluid (BALF) was collected by injecting the airways with 0.8 ml of sterile saline solution under constant pressure via a 22-gauge catheter inserted into the trachea followed by removal under constant pressure. The BALF was centrifuged to remove cells and cell particles; the supernatant was stored at −80°C for use in Western blotting. The heart and lungs from each mouse were removed en bloc. The right lung was tied off, and the left lung was inflated with 1% low-melt agarose in PBS with constant pressure (25 cm H2O) and placed on ice as we have described previously [10, 20]. The right lung was removed and frozen in liquid nitrogen and stored at −80°C for use in Western blotting. The heart was then removed and the agarose-inflated left lung was placed in 10% neutral buffered formalin (Thermo Fisher Scientific) at 4°C for 48–72 h. Following fixation, the left lung was processed for paraffin embedding. The heart was then dissected into the RV and left ventricle plus septum (LV+S). Both portions of the heart were then weighed and the RV and LV+S ratio determined [RV/(LV+S)].
Asp ag model of Th2-induced pulmonary vascular remodeling
Asp ag that did not contain any viable fungus (GREER Laboratories, Inc., Lenoir, NC) was diluted to 1 mg/ml in sterile saline. Mice were injected i.p. with 100 μl of this solution on days 0 and 14 of this experiment. On days 28–30, the mice were intranasally challenged with 100 μg Asp ag diluted in 50 μl of saline or with 50 μl saline alone. The intranasal challenges were also repeated on days 35–37, 41–43, and 45. On day 46, the mice were sacrificed and processed as stated above.
Chronic hypoxia model of pulmonary vascular remodeling
Mice were exposed to normal room air (20.8% O2) or 10.0% O2 for 4 or 28 days as we have described [10, 20, 21, 33–36]. The mice were housed in Plexiglas hypoxia chambers that had continuous air flow. The fractional concentration of O2 was monitored and controlled with a Pro:Ox model 350 unit (Biospherix, Redfield, NY) by infusion of N2 (Roberts Oxygen; Rockville, MD) balanced against an inward leak of air through holes in the chamber. CO2 and ammonia were scavenged throughout the experiment. At each time point, the mice were sacrificed and processed as stated above.
Assessment of pulmonary vascular remodeling
Pulmonary vascular remodeling was assessed in a blinded fashion as we have previously described [10, 20, 33–36]. For our initial evaluation, lung sections were stained with hematoxylin and eosin. To evaluate the muscularization of pulmonary vessels, we dual stained additional lung sections for von Willebrand Factor (endothelium) and α-smooth muscle actin (vascular smooth muscle) as we have previously described [10, 20, 35, 36]. We then examined approximately 100 randomly selected peripheral vessels (<80 μm in diameter) from each mouse (n ≥ 3 for each experimental group) under an Olympus-BHS microscope attached to a QImaging Retiga 4000RV digital camera. These vessels were classified as non-muscular (NM), partially muscular (PM), or fully muscular (FM) and analyzed as we have published [10, 20]. Vascular remodeling was also evaluated by the percent of medial thickness (%MT) of pulmonary vessels as described [20, 33]. Briefly, %MT was determined on the murine lung sections receiving Movat pentachrome stain and calculated as follows: %MT = [(external diameter – internal diameter)/external diameter] × 100. For these studies, we evaluated vessels that had an external diameter between 25 and 150 μm. Approximately 30 vessels in 30 consecutive fields were evaluated per lung. Pulmonary vessels were classified as either peribronchial or intra-alveolar. MetaMorph software (Molecular Devices, Downingtown, PA) was used to make the measurements.
Western blot analysis
Frozen mouse lung tissue was homogenized on ice with a Brinkman Homogenizer (Polytron, Westbury, NY) in homogenization/lysis buffer and the resultant protein assayed as we have described [20]. The lung and BALF samples were resolved by a 4–20% SDS-PAGE (Bio-Rad, Hercules, CA) gel and transferred onto nitrocellulose (Bio-Rad) membranes. The membranes were blocked with 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h and then incubated with polyclonal goat anti-mouse RELMα (1:500; R&D Systems, Minneapolis, MN) antibodies in 5% milk/TBS-T overnight at 4°C. The blots were then incubated in rabbit anti-goat IgG antibodies (1:5000; Bio-Rad) conjugated to horseradish peroxidase in 5% milk/TBS-T for 1h at room temperature. Enhanced chemiluminescence (Amersham, Piscataway, NJ) was used to develop the blots followed by exposure to X-ray film (Denville Scientific, Metuchen, NJ). To ensure equal loading and transfer, blots were stripped with Blot Restore Membrane Rejuvenation Kit (Millipore) and reprobed with murine anti-β actin (Santa Cruz Biotechnology, Santa Cruz, CA).
Paraffin blocks of murine lungs exposed to saline challenge, Ova challenge, normoxia (20.8% O2), or hypoxia (10.0% O2) were cut into 6-μm sections and placed on clean positively charged glass slides. We deparaffinized, rehydrated, and performed antigen retrieval on the slides as described [10, 20]. Endogenous peroxidases, avidin, and biotin were blocked as described [10, 20]. Nonspecific protein binding was blocked by incubating the sections in normal rabbit serum for 1 h at room temperature. Then, the sections were treated with polyclonal goat anti-mouse RELMα (1:200; R&D Systems, Minneapolis, MN) antibodies or antibody diluent alone overnight at 4°C. After being washed with PBS, the slides were treated with rabbit anti-goat biotinylated secondary antibodies and then an ABC horseradish peroxidase reagent (Vectastain Elite ABC Kit; Vector Laboratories) for 30 min each at room temperature. The HIMF signal was developed with the Peroxidase Substrate Kit DAB (Vector Laboratories), counterstained with hematoxylin, dehydrated, and mounted as we have described [10, 20]. The signal was visualized with an Olympus-BHS microscope attached to a QImaging Retiga 4000RV digital camera. Images were captured with ImagePro Plus (version 5.1) software.
Immunofluorescence microscopy
Paraffin-embedded murine lung tissue was sectioned, deparaffinized, and rehydrated; antigen retrieval was performed as described under Immunochemistry. The slides were then washed with PBS, and nonspecific protein binding was blocked by incubating the sections in normal horse serum for 1 h at room temperature. The sections were then treated with polyclonal rabbit anti-mouse RELMα (1:200; Abcam, Cambridge, MA) and mouse anti-α-smooth muscle actin (1:500), rat anti-F4/80 (1:100; Serotec, Raleigh, NC), mouse anti-CD3 (1:200; Santa Cruz Biotechnology), rat anti-major basic protein (MBP; 1:500; Santa Cruz Biotechnology), rat anti-neutrophil (NIMP-R14; 1:100; Abcam), rat anti-CD19 (1:200; Abcam), or diluents alone overnight at 4°C. Next, the sections were incubated with DyLight 488-labeled goat anti-rabbit IgG (H+L) (1:200; Jackson ImmunoResearch Labs, Inc., West Grove, PA) and either DyLight 594-labeled donkey anti-mouse IgG (H+L) (1:200; Jackson ImmunoResearch Labs, Inc.) or DyLight 594-labeled donkey anti-rat IgG (H+L) (1:200; Jackson ImmunoResearch Labs, Inc.) for 45 min at room temperature in the dark. Finally, the sections were washed in PBS, mounted with Vectashield Hardset Mounting Media with 4',6'-diamidino-2-phenylindole dilactate (DAPI; Vector Laboratories), covered with a glass coverslip, and visualized with an Olympus-BHS microscope attached to a QImaging Retiga 4000RV digital camera. Images were captured by using ImagePro Plus (version 5.1) software and colorized with Adobe Photoshop CS5 software (Adobe Systems, Inc., San Jose, CA).
Genomic analysis
RNA was extracted from lung tissue with the Trizol Reagent method (Invitrogen, Carlsbad, CA). Additional purification was performed on RNAeasy columns (Qiagen, Valencia, CA). The quality of total RNA samples was assessed by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). The RNA samples were labeled according to the chip manufacturer's recommended protocols. Briefly, 0.5 μg of total RNA from each sample was labeled by using the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX) in a two-step process of cDNA synthesis followed by in vitro RNA transcription. Single stranded RNA (cRNA) was generated and labeled by incorporating biotin-16-UTP (Roche Diagnostics GmbH, Mannheim, Germany). Seven hundred fifty nanograms of biotin-labeled cRNA was hybridized for 16 h to Illumina Sentrix Mouse Ref8_v1.1 BeadChips (Illumina, San Diego, CA). The hybridized biotinylated cRNA was detected with streptavidin-Cy3 and quantified by using Illumina's BeadStation 500GX Genetic Analysis Systems scanner. Preliminary analysis of the scanned data was performed with Illumina BeadStudio software, which returns single intensity data values/gene following the computation of a trimmed mean average for each probe type represented by a variable number of bead probes/gene on the array. Z-transformation for normalization was performed on each Illumina sample/array on a stand-alone basis [37], and significant changes in gene expression between class pairs were calculated by Z test [38]. Significant gene lists were calculated by selecting genes that satisfied a significance threshold criteria of Z test P values ≤ 0.001, a false discovery rate ≤ 0.1 [39], and a fold change ± 1.5 or greater. Changes in gene expression across multiple groups were tested for by one-way ANOVA. Gene expression signatures robustly identified on the basis of statistical significance were tested against multiple sample datasets by using a systematic gene set analytical approach such as Parametric Analysis of Gene Expression (PAGE) [40] or Gene Set Enrichment Analysis [41]. Differential regulation of related gene expression signatures were quantified and compared by using Gene Set Matrix Analysis [42], a modification of the PAGE method in which multiple gene lists were automatically tested against one or more datasets for parametric enrichment (using the log ratios of calculated changes in gene expression for the entire dataset).
Statistical analysis
Experimental values are expressed as means ± standard error of the mean (SEM). A paired Student's t-test was used to compare the mean responses between two groups. ANOVA was used to compare the mean responses between experimental and control groups for experiments containing multiple groups. The Tukey multiple comparison test was used to determine between which groups significant differences existed. A P value of <0.05 was considered statistically significant.
Induction of pulmonary vascular remodeling by chronic hypoxia and Th2 inflammation
Effect of chronic hypoxia and Th2 inflammation on indicators of PH
Mice exposed to chronic hypoxia for 28 days had an elevated mPAP (~1.75-fold) compared to normoxic controls (hypoxia: 30.29 ± 1.44 mmHg vs. normoxia: 17.23 ± 0.57 mmHg; P<0.001; Figure 1B). Further analysis revealed that animals exposed to hypoxia had a ~2.1-fold increase in RV/(LV+S) ratio (0.407 ± 0.050) compared to normoxic controls (0.192 ± 0.018; Figure 1C). In a separate experiment, mice that were challenged with intranasal Ova exhibited a slight (~1.3-fold) increase in mPAP compared to mice challenged with saline (Ova challenge: 22.65 ± 0.37 mmHg vs. saline: 17.88 ± 0.94 mmHg; P<0.01; Figure 1B). Even though mPAP increased slightly in the Ova-challenged mice, there were no changes in RV/(LV+S) ratio when compared to saline-challenged controls (Ova challenge: 0.216 ± 0.025; saline: 0.200 ± 0.013; Figure 1C). Asp ag challenge also failed to produce a change in RV/(LV+S) ratio (0.198 ± 0.012; Figure 1C).
Evaluation of chronic hypoxia- and Th2 inflammation-induced pulmonary vascular remodeling
Genomic analysis of lung tissue
Commonly upregulated genes
Potential function in PH
endothelial cell proliferation
smooth muscle cell proliferation
tissue remodeling
Tenascin C
smooth muscle cell proliferation
potential PH biomarker
tissue remodeling
mucus secretion
airway hyperreactivity
tissue remodeling
potential PH biomarker
potential PH biomarker
Cathepsin K
tissue remodeling
Evaluation of HIMF expression
Cellular localization of HIMF
The current study demonstrates that both chronic hypoxia and repeated airway antigen challenges induce pulmonary vascular remodeling in adult male C57BL/6 mice; however, differences were apparent in the types and extent of remodeling. Exposure to chronic hypoxia for 28 days increased all of the indicators of PH that we examined in this study [mPAP, RV/(LV+S) ratio, vascular remodeling]. In contrast to Ova-challenged mice, hypoxic mice exhibited marked increases in both mPAP and RV/(LV+S) ratio. In addition, mice exposed to chronic hypoxia exhibited an overall neo-muscularization of the pulmonary vessels throughout the entire lung in which both intra-alveolar and peribronchial vessels were thickened. The vessels from this model had the appearance of concentric remodeling. At the 28-day time point, there was little apparent perivascular inflammation. Several recent reports have suggested that Th2 inflammation is involved in the pulmonary vascular remodeling associated with the development of PH [6, 13, 15–18, 59]; therefore, we employed extended Ova-and Asp ag-challenge models (3 challenges/week, 4 weeks) to ellicit this response. Analysis of the parameters for PH revealed a slight, but statistically significant increase in mPAP (~1.3-fold) compared to saline control but no change in RV/(LV+S) ratio compared to saline-challenged mice. As expected, extended intranasal Ova challenges produced large amounts of pulmonary inflammation, particularly around the peribronchial vessels, but little new muscularization of peripheral pulmonary vessels; specific analysis of the medial thickness of both intra-alveolar and peribronchial vessels revealed extensive hypertrophy/hyperplasia in only the peribronchial vessels. The remodeling that we observed in peribronchial vessels looked very different from remodeled vessels in the hypoxic mice; the vessels from Ova-challenged mice appeared to have disorganized hypertrophy/hyperplasia. Also, the vessels that appeared to be the most remodeled had the most HIMF-expressing inflammatory cells associated with them.
Several Th2 inflammatory models of pulmonary vascular remodeling have been developed in recent years (e.g., Ova-challenge, S. mansoni infection), but these models have limitations regarding the development of PH. As opposed to chronic hypoxia, in which experimental animals consistently display elevated pulmonary and RV pressures, Th2 inflammatory models display little or no pulmonary pressure change [15, 19, 60]. In the current study, chronic hypoxia induced an almost two-fold increase in both mPAP and RV/(LV+S). Although we did observe a mild increase in the mPAP (~1.3-fold increase) of our Ova-challenged mice, this change was not accompanied by RV hypertrophy. Daley et al.[15] reported no changes in right ventricular end systolic pressure in association with antigen challenge. Crosby et al.[60] reported extensive pulmonary vascular remodeling associated with a murine model of S. mansoni infection, but these animals did not experience significant PH; the authors did suggest that mPAP was higher in those mice that had a greater and more widespread egg burden. In another study on S. mansoni infection, Graham et al.[19] also reported extensive remodeling, but demonstrated significant RV pressure increases only in IL-13Rα2 knockout mice, not in infected wild-type mice. It is possible that elevated pulmonary pressures were not observed in these models because of the time frames used for these experiments. A follow-up study performed by Crosby et al., [61], demonstrated that S. mansoni infection induced pulmonary vascular remodeling, increased RV pressures, as well as RV hypertrophy at 25 weeks following initial infection. These investigators also demonstrated that the severity of the vascular remodeling correlated with the proximity of the S. mansoni eggs. Another long-term study performed by Mushaben et al., [62] demonstrated that chronic exposure to house dust mite antigen induced pulmonary vascular remodeling at 7 and 20 weeks of exposures. This study also demonstrated increased RV pressures at 20 weeks, but not at 7 weeks. These studies suggest that it may be possible that a chronic Th2 stimulus may be required for the development of PH in these models.
Immunohistochemical expression patterns of HIMF following either chronic hypoxia or Ova challenge revealed a marked difference between these two models. Most significant among these differences was the consistent expression of HIMF in the vascular endothelium and smooth muscle throughout the lung following chronic hypoxia and the complete absence of HIMF expression in the vasculature following Ova challenge. Hypoxia induced HIMF staining in airway epithelial cells, alveolar type II cells, endothelium, vascular smooth muscle, and inflammatory cells, findings consistent with data that we have previously reported in both mice and rats [20, 21] as well as human PH lung [31]. Using double-labeling immunofluorescence microscopy, we also showed that HIMF co-localizes with smooth muscle cells only in the chronic hypoxia model. It is likely that a hypoxic stimulus induces smooth muscle cells within the lung to directly express HIMF, which could initiate the vascular remodeling process. This finding is consistent with our prior work in which we showed that knockdown of HIMF reduced chronic hypoxia–induced vascular remodeling [20]. We did observe that HIMF was expressed in macrophages, T-cells, and neutrophils during chronic hypoxia, but these co-localization events occurred less consistently than did HIMF co-localization with α-smooth muscle actin. After Ova challenge, HIMF was exclusively expressed in airway epithelial cells and inflammatory cells, a finding consistent with those of previous studies that used Ova or Asp ag challenge [15, 23, 27]. The initial study by Holcomb et al.[23] demonstrated HIMF expression in alveolar type II cells following aerosolized Ova challenge in adult female BALB/c mice, but no expression in the vascular tissue. The expression pattern of HIMF is very similar in another Th2-dependent model of pulmonary vascular remodeling. Following S. mansoni infection, HIMF is also upregulated in areas surrounding the typical granulomas, but not in the remodeled vessels themselves [19]. The lack of HIMF expression in the vascular tissue during inflammation-induced remodeling would suggest that HIMF may be acting on the vasculature through an indirect process. To examine this concept, we identified the inflammatory cells that expressed HIMF after Ova challenge. We found that HIMF was expressed by several different inflammatory cell types, including macrophages, T-cells, and neutrophils, in Ova-challenged mice and that very large numbers of such cells surrounded the remodeling peribronchial blood vessels. The expression of HIMF by macrophages is consistent with previously reported data [23]. To the best of our knowledge, this is the first study to demonstrate that HIMF is expressed in neutrophils and T-cells at the site of inflammation within the lung. At the time point examined, HIMF was not expressed in eosinophils or B-cells. It is possible that HIMF is expressed by these cells [63] at a different time point or in a different mouse strain than those used for this study. We have previously demonstrated that the human homolog to HIMF, RELMβ, is expressed in macrophages and T-cells as well as in vascular cells, plexiform lesions, and myofibroblasts, in patients diagnosed with scleroderma-associated PH [31].
The time course of HIMF expression also differs following hypoxia and antigen challenge. During chronic hypoxia, HIMF mRNA expression is maximal 24 h after initial hypoxic exposure [21]; HIMF protein expression peaks at 4 days of chronic hypoxia and gradually decreases until it reaches baseline levels at approximately 14 days [21]. In the mouse model, the increased HIMF expression correlates with the hyperplastic phase of lung vascular remodeling. This is also consistent with our observations that HIMF is upregulated in the vascular smooth muscle cells that are undergoing cell division [proliferating cell nuclear antigen (PCNA)- and/or Ki67-positive]. Other work from our laboratory supports this hyperplastic role for HIMF, as we have found HIMF to exert a marked shift in human mesenchymal cells to a dividing phenotype [64]. Because HIMF is primarily expressed during the early hyperplastic phase of chronic hypoxia and we have demonstrated that preventing such expression prevents long-term remodeling associated with chronic hypoxia [20], we hypothesize that HIMF acts as an initiating factor for pulmonary vascular remodeling. Lack of HIMF protein within the lungs at this early point of chronic hypoxia reduces the extensive remodeling that would normally result [20]. With Ova challenge, HIMF appears to be consistently upregulated throughout the time period of the model, albeit focal to the area of inflammation. Sun et al.[27] has reported increased HIMF expression following an initial 7-day Ova challenge. We and others have demonstrated extensive HIMF expression following extended Ova challenges (3 challenges/week, 4 weeks) [15, 22, 23, 27].
It is currently unknown whether HIMF gene expression is regulated differently through hypoxia and Th2 stimulation or if changes in the two models are parallel. Evidence from sequence analysis and other published work suggests that the transcription factors CCAAT box enhancer binding protein-β (C/EBPβ), hypoxia-inducible factor-1α (HIF-1α), signal transducers and activators of transcription-6 (STAT6), and/or NF-κB may be involved in HIMF gene regulation [21, 24, 26, 65–68]. Stutz et al.[26] demonstrated that HIMF itself is activated by the Th2 inflammatory pathway in cultured murine bone marrow cells via IL-4 and IL-13 activation of STAT6 and/or C/EBPβ in its promoter region. Another previous study demonstrated that bleomycin-induced pulmonary HIMF mRNA expression was partially blocked in IL-4 and IL-13 knockout mice [24]. This same study also demonstrated that combined knockout of IL-4 and IL-13 completely blocked HIMF expression in the bleomycin-injured lung. Also, the number of HIMF-positive cells located around remodeled vessels in an Asp ag-challenge model was reduced in IL-4 knockout mice compared to that in wild-type controls [15]. In S. mansoni infection, HIMF expression is at least partly dependent on IL-13Rα1 [19]. Currently, not much is known about hypoxia-induced regulation of HIMF. In a recent study published by our laboratory, we demonstrated hypoxia-induced HIMF expression in both IL-4 and STAT6 knockout mice [14]. This finding would suggest that Th2 inflammation and hypoxia regulate HIMF expression through different mechanisms. This possibility is consistent with both hypoxia-related and Th2-related transcription factor binding sites in the regulatory regions of the HIMF gene.
We have previously shown that HIMF has mitogenic, angiogenic, anti-apoptotic, vasoconstrictive, and chemokine-like properties both in vivo and in vitro[10, 20, 21, 28, 29, 68]. These are key processes involved in the pathogenesis of PH. We have demonstrated that the addition of recombinant HIMF induces a dose-dependent growth response of cultured pulmonary vascular smooth muscle cells [21], and others have shown that intratracheal instillation of HIMF induces proliferation of pulmonary vascular smooth muscle in vivo[69]. The addition of recombinant HIMF in vitro activates the phosphoinositide 3-kinase (PI-3K) as well as the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways [21, 65–67, 70–72], both of which are key proliferative pathways. These signaling events occur in isolated primary cultured rodent and human pulmonary vascular smooth muscle cells, illustrating that HIMF acts directly on the cultured cells to induce proliferation; this signaling appears to occur independently of any secondary inflammatory effects that could be induced by HIMF. The differential expression that we have observed may affect the pulmonary vascular remodeling that is observed in each model. It is possible that upregulation of HIMF in both vascular smooth muscle and endothelium may contribute to an autocrine pathway in which HIMF induces cell proliferation within the vasculature. The overexpression of HIMF in the lung vasculature appears to contribute to the overall vascular remodeling of the lung that we are seeing during chronic hypoxia. We have previously shown that AAV-HIMF–induced overexpression of HIMF in the pulmonary vasculature leads to proliferation of both vascular smooth muscle and endothelial cells [20]. During Ova challenge, pulmonary vascular remodeling is localized to the vessels associated with large airways. In this model, large amounts of HIMF are produced and secreted by airway epithelial and inflammatory cells, but no HIMF is expressed in the pulmonary vasculature itself. It is reasonable to suggest that the HIMF secreted into the surrounding areas of the lung affects the local pulmonary vessels and thereby drives the localized remodeling. Daley et al.[15] reported that in an Asp ag-challenge model, HIMF was expressed in areas surrounding remodeled vessels similar to what we have observed in our Ova-challenge model. The smooth muscle cells in these remodeled vessels also express the markers for cellular proliferation, Ki67 and PCNA [15].
Our results demonstrate that pulmonary vascular remodeling in mice induced by chronic hypoxia or antigen sensitization and challenge is associated with marked increases in HIMF expression both at the message and protein levels. HIMF was upregulated in the airway epithelium and inflammatory cells in both models, although it was expressed in the vascular smooth muscle of the hypoxia model only. It is certainly possible that the expression of HIMF within the pulmonary vessels themselves may lead to more robust vascular remodeling; therefore, leading to increased pulmonary pressure and right heart hypertrophy. Taken together, our data suggest that HIMF is a key factor in the etiology of both hypoxia- and inflammation-induced models of pulmonary vascular remodeling.
Pulmonary hypertension
- mPAP:
Mean pulmonary artery pressure
Human immunodeficiency virus
Chronic obstructive pulmonary disease
Pulmonary vascular resistance
Right ventricle
Left ventricle
- Th1:
T-helper 1
- Th2:
T-helper 2
- Ova:
- Asp ag :
Aspergillus fumigatus antigen
- S. mansoni :
Shistosoma mansoni
Hypoxia-induced mitogenic factor
Found in inflammatory zone 1
- RELMα:
Resistin-like molecule alpha
- RELMβ:
Resistin-like molecule beta
Stromal cell-derived factor-1
Monocyte chemoattractant protein-1
Human herpes virus 8
Bronchalveolar lavage fluid
Hematoxylin & eosin
% of medial thickness
Horseradish peroxidase
Tris-buffered saline with 0.1% Tween 20
Standard error of mean
Proliferating cell nuclear antigen
4',6'-diamidino-2-phenylindole dilactate
Gene Set Matrix Analysis
- C/EBPβ:
CCAAT box enhancer binding protein-β
- HIF-1α:
Hypoxia-inducible factor-1α
Signal transducers and activators of transcription-6
Phosphoinositide 3-kinase
Extracellular signal-regulated kinase.
This work was supported by NIH SCCOR P50 084946 (RAJ), NIH NHLBI R01 39706 (RAJ), and NIH fellowship T32 GM075774-09 (DJA). The authors thank Ms. Claire Levine for critical review and assistance with the manuscript preparation. We also thank Ms. Dea Sloan, Ms. Diana Zhu, and Mr. Noah Weiner for technical assistance.
Authors' Affiliations
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subset: commoncrawl


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- 1a style of cooking Italian cuisine See related entries: Dining out
- 2the food served in a restaurant (usually an expensive one) The hotel restaurant is noted for its excellent cuisine. See related entries: Dining out see also haute cuisine, nouvelle cuisine Wordfinderà la carte, course, cuisine, menu, order, reservation, restaurant, service charge, speciality, waiter Word Origin late 18th cent.: French, literally
BrE BrE//kwɪˈziːn//; NAmE NAmE//kwɪˈziːn//[uncountable, countable] (from French) Dining out
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South Sudan Plans Unity State Oil Restart, Refinery by JulyMading Ngor
South Sudan plans to resume crude output in Unity state by July after conflict in the world's newest nation caused the northern region to freeze production.
Oil fields in Unity will gradually raise output toward the 50,000 barrels per day they produced before the December shutdown, Petroleum Ministry spokesman Nicodemus Ajak Bior said in an interview today in the capital, Juba. A new refinery built by Russian and South Sudanese companies near the state capital, Bentiu, will begin producing 3,000 barrels per day of diesel in July, he said.
"In the beginning there will be a challenge to bring back production to the pre-shutdown levels," Bior said. "People are working day and night to see to it that production has restarted."
South Sudan's oil output has fallen by about a third since fighting erupted on Dec. 15 between factions loyal to President Salva Kiir and his former deputy Riek Machar. Violence has left thousands of people dead and forced more than a million to flee their homes, according to the United Nations.
The country is currently producing about 160,000 barrels per day from Upper Nile, the only state still pumping crude, Bior said. Machar has vowed to seize key oil installations in a bid to starve the military of revenue.
Government forces retook Bentiu from rebels on Jan. 26 and the Juba-based Sudd Petroleum Operating Co. has assessed damage to the facilities, Bior said. China National Petroleum Corp., India's Oil & Natural Gas Corp. and Petroliam Nasional Bhd., the main producers of South Sudan's oil, evacuated employees from the country due to the fighting.
Refining Diesel
Construction is finished on Bentiu's refinery, a joint venture by Russia's Safinat and the state-owned Nile Petroleum Corp., Bior said. A later expansion will raise output to 5,000 barrels per day, he said, without specifying a timescale.
"The refinery is ready, however commissioning will commence once the oil field in Unity state resumes production," Bior said.
Construction of a 10,000 barrel-a-day refinery in Melut county, Upper Nile state, has halted due to the conflict, Bior said. Texas-based Ventech Engineers International LLC was building the facility which is set to produce diesel, kerosene and fuel oil, he said.
South Sudan, which gained independence from Sudan in July 2011, has sub-Saharan Africa's third-biggest oil reserves, according to BP Plc data.
The country's low-sulfur crude is prized by Japanese buyers as a cleaner-burning fuel for power generation. The country has the capacity to produce as much as 350,000 to 400,000 barrels per day, Foreign Minister Barnaba Marial Benjamin said on Feb. 11.


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Workshop for Art Lovers, Teachers, Group Leaders: "Girl with a Pearl Earring"
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Johannes Vermeer, Girl with a Pearl Earring, ca. 1665. Oil on canvas. Royal Picture Gallery Mauritshuis, The Hague. Bequest of Arnoldus des Tombe, 1903 (inv. no. 670). Image courtesy of the Royal Picture Gallery Mauritshuis, The Hague
Artist-teacher Susan Hyde Greene will lead participants in hands-on experiences that explore Girl with a Pearl Earring. Adaptable to all skill levels, the activities can enhance individual appreciation and can be used in classrooms. The workshop is $25 ($10 for museum members) and includes materials and a docent-led tour.
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Healthy circulation and blood flow are important for a healthy heart The natural amino acid Arginine can act as a powerful vasodilator, helping to maintain blood flow to the heart and a healthy vascular system Arginine also has a positive effect on muscle and is involved in red blood cell formation Herpes simplex (cold sore) sufferers are advised to consult a nutritionist before taking this product
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News About Tulloch Stakes betting
Atlante A Narrow Favourite In 2014 Tulloch Stakes Betting Markets
Atlante is currently a narrow favourite in 2014 Tulloch Stakes betting market; with the Fastnet Rock colt available at odds of $4 to record his maiden win in Australia in the Group 2 event this weekend.
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Taking place as part of the bumper Golden Slipper day card at Rosehill on Saturday, the ...
Close Top End of 2013 Tulloch Stakes Betting Market
There is little separating the top three rated chances in the Group 2 2013 Tulloch Stakes (2000m) which will take place at Rosehill on Saturday.
is one of the top chances in the Tulloch Stakes. Photo: Steve Hart."]Current favourite to win the three-year-old race is Darley's Proverb who is at $4 despite having no significant lead-up results.
The winner of ...


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Mountain Shadow Transportation Group promises competitiverates and providing quality service for your company. In this recessive marketretaining services that saves companies, plays an important impact to yourbottom line. Perfect & Professional Service Guaranteed!
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7 Weight Loss Tips from The Biggest Loser
The holidays don't have to be hard on your waistline. We asked past winners of NBC's The Biggest Loser how they keep the weight off—even when the snow is piled deep and the buffet is piled high.
1. Don't let the weather stop you.
"The winter was freaking me out—I was afraid I wouldn't be able to exercise. So I'm learning to play ice hockey! To keep yourself going, find things that are fun for you."
—Helen Phillips, season 7
2. Get back up after you fall.
"I lost 214 pounds on The Biggest Loser, but eventually the weight started to creep back on because I stopped making the effort. I had to remember: Going to the gym is not a punishment—it's something I do because I care about myself."
—Erik Chopin, season 3
3. Want it? Have it! (A little.)
"My strategy when it comes to cravings: Eat what's plaguing you, but just a small portion. Otherwise, you eat around the craving. And if you add up the calories, it'll probably be more than if you had just eaten what you wanted."
—Erik Chopin
4. Make it easy to measure.
"All my serving utensils are measured. It's not obvious, because they're really beautiful, but I have the option of tracking what I'm eating. That helps me be smart about it."
—Ali Vincent, season 5
5. Remember that age is just a number.
"Before I went on the show, I sat on the couch and lived my life vicariously through the TV. Now, in addition to ice hockey, I've tried kayaking and I've become a runner—and I'm 50 years old! If you want to lose the weight, ask yourself, Is it that I can't or that I won't?"
—Helen Phillips
6. Set small goals, and celebrate when you reach them.
"I needed targets, different things to go for on a daily basis—a distance on the treadmill or a weight goal. Without them, I wasn't celebrating myself enough, and I got really good at beating myself up."
—Ali Vincent
7. Squeeze it in.
"When I'm in the grocery store, I'll do lunges up and down the aisles. In the checkout line, you could do squats. I used to worry about what people thought of me, but I don't care anymore. I know I'm going to get the last laugh."
—Ali Vincent
Thanks! You're on our list.
And about to get a whole lot healthier.
Some people like to travel by train because it combines the slowness of a car with the cramped public exposure of an airplane.
Dennis Miller
I think my pilot was a little inexperienced. We were sitting on the runway, and he said, "OK, folks, we're gonna be taking off in a just few—whoa! Here we go."
Kevin Nealon
"I can't wait until your vacation is over." —Everyone following you on Instagram
A man knocked on my door and asked for a donation toward the local swimming pool. So I gave him a glass of water.
Comedian Greg Davies
Just found the worst page in the entire dictionary. What I saw was disgraceful, disgusting, dishonest, and disingenuous.
Client: We need you to log in to the YouTube and make all our company videos viral.
From clientsfromhell.net
My cat just walked up to the paper shredder and said, "Teach me everything you know."
"Just because you can't dance doesn't mean you shouldn't dance." —Alcohol
@yoyoha (Josh Hara)
My parents didn't want to move to Florida, but they turned 60 and that's the law.
—Jerry Seinfeld
Q: What do you call an Amish guy with his hand in a horse's mouth?
A: A mechanic.


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I walked with Jenna over to Colt as he turned to smile at us. "Hey g- Bailey what happened to your face?" He gently took my face in his hand turning my face to the side moving my hair away from my face.
© Copyright 2017 FutureMarinesGirl. All rights reserved.
Status: Finished
Genre: Young Adult
omigosh that chapter was so sad!
jayden is so mean! what happened to him?
poor bailey!
update me?
OMG that chapter was awesome..
ommmg thats awesome. kmu. was it the cancer??? ahhhh. AWESOME! and jayden is an ass! bailey was just standing up for herself when sophie called her a prostitute.
why did he just insulted her...
even though he didnt 'like' her...
he doesnt have the rights to insult her what a jerk...
I hope she's fine and that someone would find her and that jerk feel very very guilty....
lol im so dramatic...
GREAT chapter though
love it....
Everyone sais what happened to Jay? LOL Sophia happened
I can't believe Jayden actually got with her though.
Sophia needs to go die a horrible death and Jayden needs to get a major reality check or join her.
Love it Girl!!!!!!
You're Awesomeness!
omg update now! what the hell happened???
I think I hate Jayden! KMU?
Great two chapters. I was thinking she should just beat the crap out of Jay and go on a date with the twins lol
great chapter. :P stupid jeydon X( OMG Bailey! update me soon please
awww, that so sad!JAYDEN NEEDS TO GET THE FUCK OVER HIMSELF! god im mad at him >:< i hope she'll be ok. her dog is awesome! keep me posted great ch and update soon
Great chapter! Let's see . . . Jay needs his ass kicked. Sophi/biotch needs a smack down and the puppy rocks!!
woow did he really just say that?! It's not her fault she's sick, he needs to dump Sophie because it's making him a dusch bag and honestly just apologize sooo much to Bailey.
Bailey?!!!!! No!!!!
PATTY!!!!!!! Nuh uh you can not stop there! Oh and I'm starting to hate Jayden even more now. I hate hate hate him and I'm very angry with you for that ending. :(
Omg Jayden is such a douche bag... He's suppose to be her friend and tht was definitually not a friend move he pulled right there... Great chapter though and thankyou so much for the update I really appreciate it :) please please please KMU if possible... I want to know what happens now that she's passed out... :D keep up the good work lol
KMU x
Dang, I cried when Jayden said that stuff to Bailey. That's wrong. She has good friends, and pretty good life for her sickness. He's not such a great friend however! :( Poor Bails. KMU!
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You need insurance coverage! We've got it! Whether it's to protect your home, auto business, loved ones or a variety of other needs. At A Ace Insurance is your one-stop shop for all of your insurance needs.
Most insurance companies will offer you similar services. But as a customer, you must know why these services are offered, and how valuable each service is to you. Because these things can make such a huge difference later in life. For over 25 years, A Ace Insurance has been providing services that are dependable, world class, and extend well beyond the sales of an insurance policy.
We are a local, family-owned agency in Baton Rouge, LA. Our insurance professionals are always here for you when you need them. They will first listen to you, understand your needs. Then they will guide you through the various options, so that you understand the implications. Finally, they will help you select the right insurance for your need with the best price, coverage and an outstanding level of service.
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Prepare for the career fair by attending one of the workshops/events listed below. Also, check out these video tips from employers coming to the Startup Career Fair and review the "Making the Most of the Career Fair" tipsheet.
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current mood: cranky
It has been quite a while since I posted...WOW!
Update: I am still with Dar. WOOHOO! It has been just over 6 months. That is usually the make or break point for me. We hit a rough road, but we got over it. Our relationship is very strong. I am very happy to be with her.
I haven't gotten any further with my divorce from my husband. I'm just not feeling motivated to do much of anything as of late.
I am feeling depressed. I'm on meds, but they don't seem to be working. I have to get bloodowork done before my dr. will even consider changing my prescription. I really don't like having to rely on drugs to alter my mood. UHG! Why can't my body just do it on its own?! I also need to find a therapist to talk to.
I spoke with one of my pastor friends last week, and she said she would hook me up with a friend of hers who is a gay pastor. She says that this woman has her partner living in the parsonage with her. Our denomination claims that homosexuality is incompatible with Christianity, but I figure if she can be in a gay relationship, and still hold onto her pastoral calling, so can I. I am looking forward to talking with her. That will be a major load off my shoulders.
We (Dar, me and our 4 children, also some family and friends) are heading for the White Mountains in New Hampshire for 2 weeks starting this coming Sunday (July 20). I am excited, but I am also looking at it with a bit of trepidation. Last year, I went to Rhode island with Christina's family...While I had some help, I felt extremely stressed a great deal of the time because I had to deal with both kids most of the time I was there. Which, of course, meant that I did not have a very good time. It felt more like work than I vacation. I don't want to pawn my kids off on people all of the time, but it would be nice to have some time without them. Dar, however, just doesn't get it. I think she feels like she put in her dues, so she should be able to have fun. But, now she has the little kids. She says she feels like the little ones get dumped on her. I don't expect her to take them all of the time...But, I would appreciate some help. At least, if I stayed home, I would have a few days off because their dad would take them. Also, I could contain them in the house and the yard. Up there, I won't get many breaks and containing them is going to be stressful. Dar doesn't get why I become so stressed. She says, "I dealt with it for 15 years. The stress gets worse. You gotta learn to manage it!" Of course, when she said that I told her that I was beginning to wonder if I even wanted to go. Then she said, "It's not an excuse as far as I'm concerned." Then she basically said that I made my bed and that I have to lie in it. I know she didn't put it that way, exactly, but that it the basic jist of it. Basically, you chose to have two children, they are your responsibility, so deal with it. I agree, they are my resposibility, but when she asked me to marry her, she signed on. As far as I'm concerned, she is their mom, and has agreed to take responsibilty for them as well. So, it should be 50-50.
Uhm, I know myself. If I get little to no help while we are up there, and I am constantly chasing around my kids - I know that my stress level is going to go sky high, I'm going to have at least one anxiety attack (if not more), and I am going to pack up my car and the kids and go home - Call my ex and ask him to take them for a few days.
Sometimes, I wonder if I am really fit to even be a mom. They stress me out so much. I like kids up until they are walking and talking. Then, I just don't relate to them until they are like 5. The getting into everything stage is just not my cup of tea. They make me want to scream, and sometimes I do.
I love Dar very much. I just wish I could help her understand where I am coming from. She says that I am strong, but I really don't feel like it. I am not her...I need help, and I am not afraid to ask for it. But, I feel like crap when I ask for help because she always brings up how long she took care of two kids on her own...And, she expects me to be able to do that.
I just don't know what to do.


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In a message dated 5/13/1999 4:39:16 PM Central Daylight Time,
firstname.lastname@example.com writes:
<< Yes it does turn to a yellowish green color. But I think of it as I think
a tulip---enjoy the brilliant color in the spring. Which brings to mind a
topic==Spring Beauties.
I wouldn't part with a big stand of 'Fortunei Albopicta' in my garden, or
'Fortunei Aurea' even though they green up.Golden Anniversary is another.
Also, H. ventricosa 'Aureamaculata'. H. longipes 'Aurea'. 'Aoki'. What are
some others?
Chines sunrise,twinkels, seafire, feather boa.
Those are the few I noticed today.
To sign-off this list, send email to firstname.lastname@example.com with the
message text UNSUBSCRIBE HOSTA-OPEN


text:
Top Terms
Ledger Pop Journal - Celebrity & Showbiz News
Angelina Jolie Looking to Namibia for Help in Securing Dame Title?
Is Angelina Jolie looking to Namibia to help her secure the title of 'Dame' she was honored with recently?
A new gossip item has Jolie leaving the US and claims Angelina is in talks to become a citizen of Namibia so she can become an offical dame.
The 39-year-old actress was recently bestowed with an honorary damehood by Britain's Queen Elizabeth in her Birthday Honours, but as she is an American she cannot officially use the title.
However, the 'Maleficent' star has now been advised she could refer to herself as Dame Angelina Jolie by becoming a citizen of Namibia, which is part of the Commonwealth and she feels using the title would help with her charity work.
A source told The Sun newspaper: ''Angelina was blown away when she was made a dame. She thinks it will add gravitas to her in her humanitarian work.
''Gradually she will focus less on acting and more on world issues and this helps to detract from her Hollywood history.''
Angelina has close ties to the African country as it is where she gave birth to eight-year-old daughter Shiloh - her first biological child with fiancé Brad Pitt - in 2006. The youngster is considered a Namibian citizen and has a passport issued by the country.
The Namibian Embassy says it would be happy to consider the A-list actress for citizenship.
A representative said: ''We would welcome Angelina Jolie to make an application for citizenship, just like anyone else.''
Angelina - who also raises adopted kids Maddox, 12, Pax, 10, and Zahara, nine, and biological twins Knox and Vivienne, five, with Brad - has become a lauded public figure for her humanitarian efforts in ending sexual violence in conflict zones through the United Nations (UN).
© Bang Showbiz
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17 CFR 240.15d-4 - Reporting by Form 40-F registrants.
§ 240.15d-4 Reporting by Form 40-F registrants.
A registrant that is eligible to use Forms 40-F and 6-K and files reports in accordance therewith shall be deemed to satisfy the requirements of Regulation 15D ( §§ 240.15d-1 through 240.15d-21 of this chapter).
[ 56 FR 30075, July 1, 1991]
Title 17 published on 2015-12-04
The following are ALL rules, proposed rules, and notices (chronologically) published in the Federal Register relating to 17 CFR Part 240 after this date.


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A valid e-mail address. All e-mails from the system will be sent to this address. The e-mail address is not made public and will only be used if you wish to receive a new password or wish to receive certain news or notifications by e-mail.
In order to prevent spam and malicious users, we ask you to please provide us with your reason (however brief) for joining. This information will never be shared. Please be sure to check your spam or junk folder in case you do not locate your email confirmation. The content of this field is kept private and will not be shown publicly.


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French Door Blinds, Window Treatments & Shades
- Consider door shape and position of handles
- Pick shallow treatments that hang close to the door
- 1" wood blinds and roller shades are ideal
- Hold down brackets are good for regularly shut shades
- Choose cordless for a sleek look and easy control
French doors have decorative handles that could interfere with the operation of door blinds or shades, so you'll want to choose a window treatment that is not very deep with an outside mount. If you plan on regularly raising and lowering your shade, go cordless to avoid getting cords tangled up on the door handle while also increasing child and pet safety. If you plan on leaving the shade down, you should select the hold down brackets when customizing your blind. These are installed on the door to keep the bottom of the shade in place.
Blindsgalore® Horizontal Blinds 1" Vinyl
retail $25.37 $17.00 + an extra 10% off
Blindsgalore® Wood Alternative Blinds 2" Slats
retail $37.31 $25.00 + an extra 10% off
Blindsgalore® Roller Shades: Reminiscent Vinyl Blackout
retail $43.28 $29.00 + an extra 10% off
Certified Best for Kids
Products with this label meet or exceed safety recommendations approved by the Window Covering Manufactures Association (WCMA) and have undergone rigorous third-party testing.
Kellie Clements Simply Chic 2" Faux Wood Blinds
retail $55.22 $37.00 + an extra 20% off
Certified Best for Kids
Products with this label meet or exceed safety recommendations approved by the Window Covering Manufactures Association (WCMA) and have undergone rigorous third-party testing.
Blindsgalore® Signature Drapery Panel: Dynasty Faux Silk
retail $58.21 $39.00
Blindsgalore® Select Cellular Shades: Light Filtering
retail $61.19 $41.00 + an extra 10% off
Certified Best for Kids
Products with this label meet or exceed safety recommendations approved by the Window Covering Manufactures Association (WCMA) and have undergone rigorous third-party testing.
Blindsgalore® Signature Drapery Panel: Slub Sheer
retail $62.69 $42.00
Blindsgalore® Allure Drapery Pair: Ikat Sheer
retail $62.69 $42.00 + an extra 10% off
Blindsgalore® Natural Woven Shades
retail $65.67 $44.00 + an extra 10% off
Blindsgalore® Signature Drapery Panel: Quatrefoil Sheer
retail $74.63 $50.00
Blindsgalore® Allure Drapery Pair: Faux Linen Check
retail $74.63 $50.00 + an extra 10% off
Bali® 2" Faux Wood Blinds
retail $74.63 $50.00 + an extra 10% off
Certified Best for Kids
Products with this label meet or exceed safety recommendations approved by the Window Covering Manufactures Association (WCMA) and have undergone rigorous third-party testing.
Blindsgalore® Signature Drapery Panel: Classic Houndstooth
retail $80.60 $54.00
Levolor® Roller Shades: Light Filtering
retail $82.09 $55.00 + an extra 20% off
Blindsgalore® Allure Drapery Pair: Faux Linen
retail $85.07 $57.00 + an extra 10% off
Blindsgalore® Allure Drapery Pair: Faux Linen Fluted
retail $85.07 $57.00 + an extra 10% off
Blindsgalore® Allure Drapery Pair: Flush Stripe
retail $85.07 $57.00 + an extra 10% off
Bali® DiamondCell® Cellular Shades: Light Filtering Double Cell
retail $86.57 $58.00 + an extra 10% off
Certified Best for Kids
Products with this label meet or exceed safety recommendations approved by the Window Covering Manufactures Association (WCMA) and have undergone rigorous third-party testing.
Cellular / Honeycomb (9)
Energy-efficient, insulating. Lift and light control options.
Custom Drapery (12)
Pair with any blind or shade. High quality, custom fabrics.
Drapery Panels (33)
Create a premium, designer look. Two standard lengths available.
Faux Wood Blinds (3)
Look of real wood. Great for humid climates.
Mini Blinds (1)
Durable and economical choice. Aluminum or vinyl materials.
Roller Shades (13)
Minimalist design. Colors and patterns galore.
Roman Shades (2)
Fabric shades with drapery feel. Array of materials and patterns.
Sheer Shades (1)
Airy fabric with gentle vanes. Softly filters incoming light.
Solar Screens (4)
Blocks harmful UV rays. Preserves your view.
Wood Blinds (2)
Warm, timeless look. Durable, built to last.
Woven Wood Shades (1)
Made with renewable materials. Intriguing, textured look.
- Cellular / Honeycomb (9)
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- Mini Blinds (1)
- Roller Shades (13)
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- Woven Wood Shades (1)
- refine
Opaque / Blackout (33)
Opaque window treatments are made from fabrics or materials that block light.
Semi-Opaque (36)
Although light is substantially blocked, some vague shadows may be visible.
Semi-Sheer (16)
Light is gently filtered through the fabric; silhouettes are visible through the shade.
Sheer (7)
Fabric transmits light through the shade, allowing a clear view inside and out
- Opaque / Blackout (33)
- Semi-Opaque (36)
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- Sheer (7)
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0% Openness (1)
These solar shades blocks 99% of all incoming light to provide complete daytime and nighttime privacy.
10% Openness (4)
Greater openness for better view-through with good UV and glare reduction
14% Openness (3)
Allows in the second most light, good for mild light control and high view-through
3% Openness (4)
These mesh shades block the most light for maximum UV protection and glare reduction.
4% Openness (1)
These solar mesh shades offer excellent light reduction and UV protection.
5% Openness (4)
These mesh window shades offer strong light and UV reduction with slightly better view-through.
7% Openness (1)
These mesh roller shades offer a great combination of light control and view-through.
- 0% Openness (1)
- 10% Openness (4)
- 14% Openness (3)
- 3% Openness (4)
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- refine
Patterns (20)
Make a bold decorating statement with tonal or multi-colored patterned window treatments
Solid Plain (22)
Coordinate your decor with classic solids in a wide range of colors and fabrics
Solids (30)
A wide variety of colors and fabrics in solid tones
Sports and Teams (1)
Sports team merchandise brightens up your game room or kids room like nothing else. Large, crisp logos on truly functional window shades. Fun and function!
Stripes (8)
Choose subtle tonal variations or multi-colored stripes to spice up your decor
Tone on Tone (7)
Choose two alternating colors on your shade for unique decorating possibilities
Weaves (3)
Features woven fabrics, including linen and grass-like materials
- Patterns (20)
- Solid Plain (22)
- Solids (30)
- Sports and Teams (1)
- Stripes (8)
- Tone on Tone (7)
- Weaves (3)
- refine
Double Cell (6)
Multi-cell construction provides excellent insulation
Single Cell (6)
Provides great insulation in a wide variety of pleat sizes
- Double Cell (6)
- Single Cell (6)
- refine
3/8" Pleat (5)
.375 inch (3/8 of an inch), are the thinnest available, and equivalent to 32 pleats per foot.
1/2" Pleat (4)
.5 inch (1/2 of an inch), are small-medium, and equivalent to 24 pleats per foot.
9/16" Pleat (1)
.5625 inch (5/8 of an inch), are the thickness of a deck of cards and equivalent to 21 pleats per foot.
3/4" Pleat (2)
.75 inch (3/4 of an inch), are the second largest available, and equivalent to 16 pleats per foot.
- 3/8" Pleat (5)
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- 9/16" Pleat (1)
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- refine
Blackout (33)
Blackout blinds and shades can eliminate glare on TVs and monitors, make sleeping easier or simply used to darken a room.
Cordless (28)
Cordless lift option easily raises and lowers the blind or shade for convenient and safe operation
Cordless Top Down/Bottom Up (9)
Cordless top down bottom up shades allows you to lower the shade from the top, raise it from the bottom, or both.
Easy Cleaning (10)
Privacy (16)
Take a look at our wide selection of privacy window treatments.
Remote Control / Motorized (20)
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Routeless (3)
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Top down bottom up is ultimate privacy and light control option - raise the shade from the bottom, the top, or both
Vertical Blind Alternatives (2)
Unique visual appeal. Ideal for big windows or doors.
- Blackout (33)
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- Cordless Top Down/Bottom Up (9)
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- Hunter Douglas Comparable (3)
- Privacy (16)
- Remote Control / Motorized (20)
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- Top Down/Bottom Up (10)
- Vertical Blind Alternatives (2)
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Bali (14)
Since 1939, Bali has been providing beautiful, quality window treatments to customers around the world. Choose from a comprehensive selection of Bali blinds and shades.
Blindsgalore (50)
Save money without sacrificing quality. Choose from Blindsgalore's signature line of custom window treatments and coverings.
Comfortex (7)
Comfortex has developed a wide array of innovative blinds and shades to meet your needs.
Graber (1)
In business since 1939, the Graber brand has long been synonymous with style and sophistication, providing a timeless look that's always on trend.
Levolor (5)
Levolor, one of the most recognized names in custom blinds and shades, has set the standard in window fashion, design innovation and product quality since 1914.
Simply Chic (4)
Simply Chic window shades provide a custom designer look at an affordable price.
- Bali (14)
- Blindsgalore (50)
- Comfortex (7)
- Graber (1)
- Levolor (5)
- Simply Chic (4)
- refine
Free Cordless Upgrade (4)
Save big on your next blinds or shades order with a FREE upgrade to Cordless lift on these select products.
New Additions (28)
Check out the new additions to Blindsgalore's selection of custom window treatments
Top Seller (17)
- Free Cordless Upgrade (4)
- New Additions (28)
- Top Seller (17)
- refine
Horizontal (9)
Horizontal blinds and shades operate up and down instead of side to side. These types of blinds work well on nearly all windows except sliding doors.
Horizontal Blinds (5)
Horizontal blinds are any blinds that have horizontal slats that can be rotated open or closed blinds, faux wood blinds and aluminum or mini blinds.
- Horizontal (9)
- Horizontal Blinds (5)
- refine
Beige / Neutral (63)
Beige / neutral is a versatile color that goes well with many different looks.
Black (30)
Try black window treatments for a striking contrast and modern look
Blue (52)
Choose from our great selection of window treatments featuring materials and fabrics in numerous shades of blue
Brown (57)
Choose from our selection of window treatments featuring brown fabrics and materials.
Gray (51)
Choose from light gray tones to deep slate colors - everything between white and black
Green (53)
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Multi-colored (14)
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Choose from our selection of window treatments featuring orange fabrics and materials.
Purple (31)
Choose from our selection of window treatments featuring purple fabrics and materials.
Red / Pink (43)
Choose from our selection of window treatments featuring red or pink fabrics and materials
Silver (24)
Take a look at our selection of silver window treatments.
White / Off-White (67)
Choose from our selection of window treatments featuring white and off-white fabrics and materials.
Woodgrain (5)
Choose from our selection of blinds and shutters featuring real or faux woodgrain
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Choose from our selection of window treatments featuring yellow and cream colored fabrics and materials.
- Beige / Neutral (63)
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- Purple (31)
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- Silver (24)
- White / Off-White (67)
- Woodgrain (5)
- Yellow / Cream (51)
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Bamboo (1)
Flattened pieces of bamboo in various colors are the star of these window treatments
Cellular (9)
Take a look at our cellular window treatments.
Fabric (69)
Offering a variety of natural textures, fabric is featured in a number of shades and vertical blinds.
Faux Wood (3)
The look of real wood with added durability at a lower price. Great for areas of high humidity. Available in a range of colors and wood tones.
Matchsticks (1)
Whether a simple design or a complex weave, matchsticks make the shade.
PVC/Vinyl (3)
State-of-the-art material will not warp, shrink, chip or fade and is waterproof
Solar Screen (4)
Sun control shades are a simple, clean way to upgrade a window and block sunlight.
Wood (2)
Wood is the classic natural material and is available in a variety products, colors and sizes to fit your rooms needs.
- Bamboo (1)
- Cellular (9)
- Fabric (69)
- Faux Wood (3)
- Matchsticks (1)
- PVC/Vinyl (3)
- Solar Screen (4)
- Wood (2)
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Eco-Friendly (4)
Features products manufactured to the highest standards of environmental responsibility for a no-compromise upgrade.
Energy Efficient (20)
Energy efficient window coverings help lower your utility bill and while making your home more beautiful.
- Eco-Friendly (4)
- Energy Efficient (20)
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Child Safe (27)
Blindsgalore offers a wide variety of window treatment options that improve child safety.
Flame Resistant (4)
Choose from our selection of blinds and shades made with fire retardant fabrics
- Child Safe (27)
- Flame Resistant (4)
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Accordia™ (1)
Choose from a great selection of Levolor's Accordia line of energy efficient cellular shades, available in single and double cell.
CrystalPleat® (1)
Enjoy the highest level of insulation of all window treatments with Graber CrystalPleat cellular shades
Designer Series (7)
Take a look at our Blindsgalore Designer Series roman shades, drapery, and valances, offering elegance, quality and style for less.
DiamondCell™ (2)
Bali DiamondCell cellular shades offer fashion-forward window coverings at the right price
Envision™ (5)
When it's fabric that matters, the Comfortex Envision line of roller and solar shades are the window treatments you want
Gallery (1)
Northern Heights™ (1)
The Northern Heights collection is Bali's premiere line of hardwood blinds
Premium Woods (1)
Check out Levolor's Premium Woods collection of wood blind.
Select Cellular (1)
SlumberShade™ (1)
Take a look at our new sleep shades from Comfortex Window Fashions.
Wood Images® (1)
Wood Images capture the essence and look of real wood blinds, while the moisture resistant slats are perfect for high-humidity areas
- Accordia™ (1)
- CrystalPleat® (1)
- Designer Series (7)
- DiamondCell™ (2)
- Envision™ (5)
- Gallery (1)
- Northern Heights™ (1)
- Premium Woods (1)
- Select Cellular (1)
- SlumberShade™ (1)
- Wood Images® (1)
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customer reviews for french doors
"Quality at a great price"
By Denice from California
Purchased Daybreak II - 3/8" Cell Butter Creme top down bottom up to replace expensive motorized Hunter Douglas cellulars that kept malfunctioning. No regrets.View this product
"Great value for money!"
By amberdana from Becker MN
I spent a lot of time shopping for custom drapes for my two big picture windows; it was impossible to find off the rack drapes wide enough, and custom drapes through Costco's partner company were insanely expensive. I am so happy I found Blindsgalore--the employees I telephoned and emailed were so professional, and I am so happy with how great my drapes look!View this product


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Welcome to Camp Echo, Fishing, Biking, Boating and Kayaking
- Airport Shuttle
- Breakfast
- Gym
- Parking
- Pets Allowed
- Swimming Pool
- Wifi
- Smoking Allowed
Fishing, Biking, Boating and Kayaking or just relaxing, your choice.
This vacation rental has a dock on Henderson Lake in Inverness, a large fresh water lake perfect for boating and kayaking. For you biking enthusiasts the Withlacoochee Rails to Trails is just a 15 minute (bike) ride from this cute and comfortable 2 bedroom 1 bathroom house. This trail stretches 46 miles thru Citrus County and is suited for both amateur and professional riders.
Inverness is the perfect place to stay in Florida where you can get away for the day and see the Orlando theme parks, Tarpon Springs sponge docks, Crystal River Manatees. in November the Manatees come into the springs and July through Sept is Scallop season.
Travel about 30 minutes west to Crystal River and Homosassa and you have the best Gulf access in all of Central Florida.
Come and meet Homer and Pearl, nieghborhood Sandhill Cranes who visit almost daily.
Two bedrooms and one bathroom. There is a pull out queen sofa bed in the Florida Room for extra sleeping.