Placenta growth factor mediated gene regulation in sickle cell disease.

Sickle cell anemia (SCA) is an autosomal recessive disorder caused by mutation in the ß-globin gene. Pulmonary hypertension (PH), a complication of SCA, results in severe morbidity and mortality. PH is a multifactorial disease: systemic vasculopathy, pulmonary vasoconstriction, and endothelial dysfunction and remodeling. Placenta growth factor (PlGF), an angiogenic growth factor, elaborated from erythroid cells, has been shown to contribute to inflammation, pulmonary vasoconstriction and airway hyper-responsiveness (AH) in mouse models of sickle cell disease. In this review, we summarize the cell-signaling mechanism(s) by which PlGF regulates the expression of genes involved in inflammation, PH and AH in cell culture and corroborate these findings in mouse models of SCA and in individuals with SCA. The role of microRNAs (miRNAs) in the post-transcriptional regulation of these genes is presented and how these miRNAs located in their host genes are transcriptionally regulated. An understanding of the transcriptional regulation of these miRNAs provides a new therapeutic approach to ameliorate the clinical manifestations of SCA.

Activated Transcription Factor 3 in Association with Histone Deacetylase 6 Negatively Regulates MicroRNA 199a2 Transcription by Chromatin Remodeling and Reduces Endothelin-1 Expression.

Previous studies showed that high levels of placenta growth factor (PlGF) correlated with increased plasma levels of endothelin-1 (ET-1), a potent vasoconstrictor, in sickle cell disease (SCD). PlGF-mediated transcription of the ET-1 gene occurs by activation of hypoxia inducible factor 1α (HIF-1α) and posttranscriptionally by microRNA 199a2 (miR-199a2), which targets the 3' untranslated region (UTR) of HIF-1α mRNA. However, relatively less is known about how PlGF represses the expression of miR-199a2 located in the DNM3 opposite strand (DNM3os) transcription unit. Here, we show that PlGF induces the expression of activated transcription factor 3 (ATF3), which, in association with accessory proteins (c-Jun dimerization protein 2 [JDP2], ATF2, and histone deacetylase 6 [HDAC6]), as determined by proteomic analysis, binds to the DNM3os promoter. Furthermore, we show that association of HDAC6 with ATF3 at its binding site in this promoter was correlated with repression of miR-199a2 transcription, as shown by DNM3os transcription reporter and chromatin immunoprecipitation (ChIP) assays. Tubacin, an inhibitor of HDAC6, antagonized PlGF-mediated repression of DNM3os/pre-miR-199a2 transcription with a concomitant reduction in ET-1 levels in cultured endothelial cells. Analysis of lung tissues from Berkeley sickle (BK-SS) mice showed increased levels of ATF3 and increased expression of ET-1. Delivery of tubacin to BK-SS mice significantly attenuated plasma ET-1 and PlGF levels. Our studies demonstrated that ATF3 in conjunction with HDAC6 acts as a transcriptional repressor of the DNM3os/miR-199a2 locus.

Placenta growth factor augments airway hyperresponsiveness via leukotrienes and IL-13.

Airway hyperresponsiveness (AHR) affects 55%-77% of children with sickle cell disease (SCD) and occurs even in the absence of asthma. While asthma increases SCD morbidity and mortality, the mechanisms underlying the high AHR prevalence in a hemoglobinopathy remain unknown. We hypothesized that placenta growth factor (PlGF), an erythroblast-secreted factor that is elevated in SCD, mediates AHR. In allergen-exposed mice, loss of Plgf dampened AHR, reduced inflammation and eosinophilia, and decreased expression of the Th2 cytokine IL-13 and the leukotriene-synthesizing enzymes 5-lipoxygenase and leukotriene-C4-synthase. Plgf-/- mice treated with leukotrienes phenocopied the WT response to allergen exposure; conversely, anti-PlGF Ab administration in WT animals blunted the AHR. Notably, Th2-mediated STAT6 activation further increased PlGF expression from lung epithelium, eosinophils, and macrophages, creating a PlGF/leukotriene/Th2-response positive feedback loop. Similarly, we found that the Th2 response in asthma patients is associated with increased expression of PlGF and its downstream genes in respiratory epithelial cells. In an SCD mouse model, we observed increased AHR and higher leukotriene levels that were abrogated by anti-PlGF Ab or the 5-lipoxygenase inhibitor zileuton. Overall, our findings indicate that PlGF exacerbates AHR and uniquely links the leukotriene and Th2 pathways in asthma. These data also suggest that zileuton and anti-PlGF Ab could be promising therapies to reduce pulmonary morbidity in SCD.

Peroxisome proliferator-activated receptor-α-mediated transcription of miR-301a and miR-454 and their host gene SKA2 regulates endothelin-1 and PAI-1 expression in sickle cell disease.

Endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1) play important roles in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show higher levels of placenta growth factor (PlGF) in SCD correlate with increased plasma levels of ET-1, PAI-1, and other physiological markers of PH. PlGF-mediated ET-1 and PAI-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively little is understood regarding post-transcriptional regulation of PlGF-mediated expression of ET-1 and PAI-1. Herein, we show PlGF treatment of endothelial cells reduced levels of miR-301a and miR-454 from basal levels. In addition, both miRNAs targeted the 3'-UTRs of ET-1 and PAI-1 mRNAs. These results were corroborated in the mouse model of SCD [Berkeley sickle mice (BK-SS)] and in SCD subjects. Plasma levels of miR-454 in SCD subjects were significantly lower compared with unaffected controls, which correlated with higher plasma levels of both ET-1 and PAI-1. Moreover, lung tissues from BK-SS mice showed significantly reduced levels of pre-miR-301a and concomitantly higher levels of ET-1 and PAI-1. Furthermore, we show that miR-301a/miR-454 located in the spindle and kinetochore-associated protein-2 (SKA2) transcription unit was co-transcriptionally regulated by both HIF-1α and peroxisome proliferator-activated receptor-α (PPAR-α) as demonstrated by SKA2 promoter mutational analysis and ChIP. Finally we show that fenofibrate, a PPAR-α agonist, increased the expression of miR-301a/miR-454 and SKA2 in human microvascular endothelial cell line (HMEC) cells; the former were responsible for reduced expression of ET-1 and PAI-1. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-301a/miR-454 expression can ameliorate PH and lung fibrosis by reduction in ET-1 and PAI-1 levels in SCD.

Erythropoietin-mediated expression of placenta growth factor is regulated via activation of hypoxia-inducible factor-1α and post-transcriptionally by miR-214 in sickle cell disease.

Placental growth factor (PlGF) plays an important role in various pathological conditions and diseases such as inflammation, cancer, atherosclerosis and sickle cell disease (SCD). Abnormally high PlGF levels in SCD patients are associated with increased inflammation and pulmonary hypertension (PHT) and reactive airway disease; however, the transcriptional and post-transcriptional mechanisms regulating PlGF expression are not well defined. Herein, we show that treatment of human erythroid cells and colony forming units with erythropoietin (EPO) increased PlGF expression. Our studies showed EPO-mediated activation of HIF-1α led to subsequent binding of HIF-1α to hypoxia response elements (HREs) within the PlGF promoter, as demonstrated by luciferase transcription reporter assays and ChIP analysis of the endogenous gene. Additionally, we showed miR-214 post-transcriptionally regulated the expression of PlGF as demonstrated by luciferase reporter assays using wild-type (wt) and mutant PlGF-3'-UTR constructs. Furthermore, synthesis of miR-214, located in an intron of DNM3 (dynamin 3), was transcriptionally regulated by transcription factors, peroxisome proliferator-activated receptor-α (PPARα) and hypoxia-inducible factor-1α (HIF-1α). These results were corroborated in vivo wherein plasma from SCD patients and lung tissues from sickle mice showed an inverse correlation between PlGF and miR-214 levels. Finally, we observed that miR-214 expression could be induced by fenofibrate, a Food and Drug Administration (FDA) approved PPARα agonist, thus revealing a potential therapeutic approach for reduction in PlGF levels by increasing miR-214 transcription. This strategy has potential clinical implications for several pathological conditions including SCD.

MicroRNA 648 Targets ET-1 mRNA and is cotranscriptionally regulated with MICAL3 by PAX5.

Pulmonary hypertension (PHT) is associated with high mortality in sickle cell anemia (SCA). Previously, we showed that elevated levels of placenta growth factor (PlGF) in SCA patients correlate with increased levels of the potent vasoconstrictor endothelin-1 (ET-1) and PHT. Moreover, PlGF induced the expression of ET-1 via hypoxia-inducible factor 1α. Here, we show a novel example of ET-1 posttranscriptional regulation by PlGF via action of microRNA 648 (miR-648), which is subject to transcriptional coregulation with its host gene, MICAL3 (microtubule-associated monooxygenase, calponin, and LIM domain containing 3gene). PlGF repressed expression of miR-648 in endothelial cells. Luciferase reporter assays using wild-type and mutant ET-1 3' untranslated region (UTR) constructs, and transfection of miR-648 mimics showed that miR-648 targets the 3' UTR of ET-1 mRNA. Since miR-648 is located in a 5'-proximal intron of MICAL3, we examined which of three potential promoters was responsible for its expression. The MICAL3 distal promoter (P1) was the predominant promoter used for transcription of pre-miR-648, and it was under positive control by PAX5 (paired box protein 5) transcription factor, as demonstrated by the loss and gain of function of PAX5 activity, and chromatin immunoprecipitation analysis. These studies provide a novel link wherein PlGF-mediated downregulation of PAX5 attenuates miR-648 expression leading to increased ET-1 levels that are known to induce PHT in SCA.

Peroxisome proliferator-activated receptor-α-mediated transcription of miR-199a2 attenuates endothelin-1 expression via hypoxia-inducible factor-1α.

Endothelin-1, a potent vasoconstrictor, plays an important role in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show that higher levels of placenta growth factor (PlGF), secreted by erythroid precursor cells, correlate with increased plasma levels of endothelin-1 (ET-1) and other functional markers of PH in SCD. PlGF-mediated ET-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively less is understood regarding how PlGF-mediated expression of HIF-1α and its downstream effector ET-1 are post-transcriptionally regulated. Herein, we show that PlGF treatment of endothelial cells resulted in reduced levels of miR-199a2, which targeted the 3'-UTR of HIF-1α mRNA and concomitantly led to augmented ET-1 expression. Plasma levels of miR-199a2 in SCD subjects were significantly lower with reciprocally high levels of plasma ET-1, unlike unaffected controls. This observation provided a molecular link between miR-199a2 and high levels of ET-1 in SCD. Furthermore, we show that miR-199a2 located in the DNM3os transcription unit was co-transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα). Binding of the latter to PPARα cis-elements in the promoter of DNM3os was demonstrated by promoter mutational analysis and ChIP. Additionally, we show that fenofibrate, a PPARα agonist, increased the expression of miR-199a2 and DNM3os; the former was responsible for reduced expression of HIF-1α and ET-1. In vivo studies of fenofibrate-fed Berkeley sickle mice resulted in increased levels of miR-199a2 and reduced levels of ET-1 in lung tissues. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-199a2 expression can ameliorate PH by reduction of ET-1 levels.

Proteasome inhibition induces both antioxidant and hb f responses in sickle cell disease via the nrf2 pathway.

Oxidant stress is implicated in the manifestations of sickle cell disease including hemolysis and vascular occlusion. Strategies to induce antioxidant response as well as Hb F (α2γ2) have the potential to ameliorate the severity of sickle cell disease. Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2) is a transcription factor that regulates antioxidant enzymes as well as γ-globin transcription. The Nrf2 in the cytoplasm is bound to its adapter protein Keap-1 that targets Nrf2 for proteasomal degradation, thereby preventing its nuclear translocation. We examined whether inhibiting the 26S proteasome using the clinically applicable proteasome inhibitors bortezomib and MLN 9708 would promote nuclear translocation of Nrf2, and thereby induce an antioxidant response and as well as Hb F in sickle cell disease. Proteasome inhibitors induced reactive oxygen species (ROS) and thereby increased Nrf2-dependent antioxidant enzyme transcripts, elevated cellular glutathione (GSH) levels and γ-globin transcripts as well as Hb F levels in the K562 cell line and also in erythroid burst forming units (BFU-E) generated from peripheral blood mononuclear cells of sickle cell disease patients. These responses were abolished by siRNA-mediated knockdown of Nrf2. Proteasome inhibitors, especially newer oral agents such as MLN9708 have the potential to be readily translated to clinical trials in sickle cell disease with the dual end points of antioxidant response and Hb F induction.

Angiogenic growth factors augment K-Cl cotransporter expression in erythroid cells via hypoxia-inducible factor-1α.

The potassium chloride cotransporters (KCC) family of proteins are widely expressed and are involved in the transepithelial movement of potassium and chloride ions and the regulation of cell volume. KCC activity is high in reticulocytes, and contributes to the dehydration of sickle red blood cells. Because plasma levels of both vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) are elevated in sickle cell individuals, and VEGF has been shown to increase KCC expression in other cells, we hypothesized that VEGF and PlGF influence KCC expression in erythroid cells. Both VEGF and PlGF treatment of human erythroid K562 cells increased both mRNA and protein levels of KCC1, KCC3b, and KCC4. VEGF- and PlGF-mediated cellular signaling involved VEGF-R1 and downstream effectors, specifically, PI-3 kinase, p38 MAP kinase, mTOR, NADPH-oxidase, JNK kinase, and HIF-1α. VEGF and PlGF-mediated transcription of KCC3b and KCC4 involved hypoxia response element (HRE) motifs in their promoters, as demonstrated by promoter analysis, EMSA and ChiP. These results were corroborated in vivo by adenoviral-mediated overexpression of PlGF in normal mice, which led to increased expression of mKCC3 and mKCC4 in erythroid precursors. Our studies show that VEGF and PlGF regulate transcription of KCC3b and KCC4 in erythroid cells via activation of HIF-1α, independent of hypoxia. These studies provide novel therapeutic targets for regulation of cell volume in RBC precursors, and thus, amelioration of dehydration in RBCs in sickle cell disease.

15th International Symposium on Cells of the Hepatic Sinusoid, 2010.

This is a meeting report of the presentations given at the 15th International Symposium on Cells of the Hepatic Sinusoid, held in 2010. The areas covered include the contributions of the various liver cell populations to liver disease, molecular and cellular targets involved in steatohepatitis, hepatic fibrosis and cancer and regenerative medicine. In addition to a review of the science presented at the meeting, this report provides references to recent literature on the topics covered at the meeting.

Involvement of miR-30c and miR-301a in immediate induction of plasminogen activator inhibitor-1 by placental growth factor in human pulmonary endothelial cells.

PAI-1 (plasminogen activator inhibitor-1) is a key physiological inhibitor of fibrinolysis. Previously, we have reported PlGF (placental growth factor)-mediated transcriptional up-regulation of PAI-1 (SERPINE1) mRNA expression via activation of HIF-1α (hypoxia-inducible factor-1α) and AP-1 (activator protein-1) in HPMVECs (human pulmonary microvascular endothelial cells), which resulted in elevated PAI-1 in humans with SCA (sickle cell anaemia). In the present study, we have identified the role of post-transcriptional mechanism(s) of PlGF-mediated accumulation of PAI-1 mRNA in HPMVECs by examining the role of microRNAs (miRNAs/miRs) in PlGF-induced PAI-1 mRNA stability. Our results show reduced expression of miR-30c and miR-301a, but not of miR-99a, in response to PlGF, which have evolutionarily conserved binding sites in the 3'-UTR (3'-untranslated region) of PAI-1 mRNA. Transfection of anti-miR-30c or anti-miR-301a oligonucleotides resulted in increased PAI-1 mRNA levels, which were increased further with PlGF stimulation. Conversely, overexpression of pre-miR-30c or pre-miR-301a resulted in an attenuation of PlGF-induced PAI-1 mRNA and protein levels. Luciferase reporter assays using wild-type and mutant 3'-UTR constructs confirmed that the PAI-1 3'-UTR is indeed a direct target of miR-30c and miR-301a. Finally, plasma levels of miR-30c and miR-301a were significantly down-regulated in patients with SCA compared with normal controls. These results provide a post-transcriptional regulatory mechanism of PlGF-induced PAI-1 elevation.

Endothelin-1-induced macrophage inflammatory protein-1beta expression in monocytic cells involves hypoxia-inducible factor-1alpha and AP-1 and is negatively regulated by microRNA-195.

Patients with sickle cell disease (SCD) exhibit a chronic inflammatory state manifested by leukocytosis and increased circulating levels of proinflammatory cytochemokines. Our studies show that placenta growth factor levels are high in SCD, and placental growth factor induces the release of the vasoconstrictor endothelin-1 (ET-1) from pulmonary microvascular endothelial cells. In this study, we observed that ET-1 increased the expression of the chemokines MIP-1ß or CCL4. ET-1-induced MIP-1ß mRNA expression in THP-1 cells and human peripheral blood monocytes occurred via the activation of PI3K, NADPH oxidase, p38 MAPK, and JNK-1 but not JNK-2. ET-1-induced MIP-1ß expression involved hypoxia-inducible factor-1α (HIF-1α), independent of hypoxia, as demonstrated by silencing with HIF-1α small interfering RNA, EMSA, and chromatin immunoprecipitation analysis. ET-1-induced MIP-1ß promoter luciferase activity was attenuated when any of the five hypoxia-response elements, AP-1, or NF-κB binding motifs in the proximal MIP-1ß promoter (-1053/+43 bp) were mutated. Furthermore, ET-1 significantly downregulated the expression of a key microRNA, microRNA-195a, which showed a complementary binding site in the 3' untranslated region of MIP-1ß mRNA. Moreover, ET-1-induced MIP-1ß mRNA expression in either THP-1 cells or peripheral blood monocytes was reduced upon expression of microRNA-195a. Conversely, transfection of monocytes with anti-microRNA-195a oligonucleotide augmented several-fold ET-1-induced MIP-1ß expression. Taken together, these studies showed that ET-1-mediated MIP-1ß gene expression is regulated via hypoxia-response elements, AP-1, and NF-κB cis-binding elements in its promoter and negatively regulated by microRNA-195, which targets the 3' untranslated region of MIP-1ß RNA. These studies provide what we believe are new avenues, based on targets of HIF-1α and microRNAs, for ameliorating inflammation in SCD.

Ethanol-induced HO-1 and NQO1 are differentially regulated by HIF-1alpha and Nrf2 to attenuate inflammatory cytokine expression.

Oxidative stress plays an important role in alcohol-induced inflammation and liver injury. Relatively less is known about how Kupffer cells respond to oxidative stress-induced expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1) to blunt inflammation and liver injury. We showed that Kupffer cells from ethanol-fed rats and ethanol-treated rat Kupffer cells and THP-1 cells displayed increased mRNA expression of HO-1, NQO1, and hypoxia-inducible factor-1α (HIF-1α). Our studies showed that silencing with HIF-1α and JNK-1 siRNAs attenuated ethanol-mediated mRNA expression of HO-1, but not NQO1, whereas Nrf2 siRNA attenuated the mRNA expression of both HO-1 and NQO1. Additionally, JunD but not JunB formed an activator protein-1 (AP-1) oligomeric complex to augment HO-1 promoter activity. Ethanol-induced HO-1 transcription involved antioxidant response elements, hypoxia-response elements, and an AP-1 binding motif in its promoter, as demonstrated by mutation analysis of the promoter, EMSA, and ChIP. Furthermore, livers of ethanol-fed c-Jun(fl/fl) mice showed reduced levels of mRNA for HO-1 but not of NQO1 compared with ethanol-fed control rats, supporting the role of c-Jun or the AP-1 transcriptional complex in ethanol-induced HO-1 expression. Additionally, attenuation of HO-1 levels in ethanol-fed c-Jun(fl/fl) mice led to increased proinflammatory cytokine expression in the liver. These results for the first time show that ethanol regulates HO-1 and NQO1 transcription by different signaling pathways. Additionally, up-regulation of HO-1 protects the liver from excessive formation of inflammatory cytokines. These studies provide novel therapeutic targets to ameliorate alcohol induced inflammation and liver injury.

Placenta growth factor-induced early growth response 1 (Egr-1) regulates hypoxia-inducible factor-1alpha (HIF-1alpha) in endothelial cells.

Leukotrienes, the lipid inflammatory products derived from arachidonic acid, are involved in the pathogenesis of respiratory and cardiovascular diseases and reactive airway disease in sickle cell disease. Placenta growth factor (PlGF), elaborated from erythroid cells, increased the mRNA expression of 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) in human pulmonary microvascular endothelial cells. PlGF-induced both promoter activity and mRNA expression of hypoxia-inducible factor-1alpha (HIF-1alpha), which was abrogated by early growth response-1 (EGR-1) small interfering RNA. PlGF showed a temporal reciprocal relationship in the mRNA levels of EGR-1 and NAB2, the latter a repressor of Egr-1. Moreover, Nab2, but not mutant Nab2, significantly reduced promoter activity and mRNA expression of HIF-1alpha and also reduced expression of the HIF-1alpha target gene FLAP. Furthermore, overexpression of Egr-1 led to increased promoter activities for both HIF-1alpha and FLAP in the absence of PlGF. Additionally, the Egr-1-mediated induction of HIF-1alpha and FLAP promoters was reduced to basal levels by EGR-1 small interfering RNA. The binding of Egr-1 to HIF-1alpha promoter was corroborated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay, which showed increased Egr-1 binding to the HIF-1alpha promoter in response to PlGF stimulation. These studies provide a novel mechanism for PlGF-mediated regulation of HIF-1alpha via Egr-1, which results in increased FLAP expression. This study provides a new therapeutic target, namely Egr-1, for attenuation of elevated leukotriene levels in patients with sickle cell disease and other inflammatory diseases.

Placenta growth factor (PlGF), a novel inducer of plasminogen activator inhibitor-1 (PAI-1) in sickle cell disease (SCD).

Sickle cell disease (SCD) is characterized by a prothrombotic state. Plasminogen activator inhibitor-1 (PAI-1) is known to modulate fibrinolysis, lung injury/fibrosis, and angiogenesis. However, its role in SCD is less understood, and the molecular mechanisms underlying increased PAI-1 are unknown. Herein, we show a novel link between PAI-1 and sickle erythropoiesis. Plasma PAI-1 levels were high in SCD patients at steady state and in two humanized sickle mouse models, with increased PAI-1 immunolabeling in sickle mouse lung, bronchial epithelial cells, alveolar macrophages, and pulmonary microvascular endothelial cells. Placenta growth factor (PlGF), released at high levels by sickle erythroblasts, induced PAI-1 expression in primary human pulmonary microvascular endothelial cells and monocytes through activation of c-Jun N-terminal kinase (JNK), NADPH oxidase, and hypoxia-inducible factor-1alpha (HIF-1alpha). Analysis of the human PAI-1 promoter revealed this induction was mediated by hypoxia-response element (HRE)-1, HRE-2, and distal activator protein (AP-1) sites. We also identify the involvement of c-Jun, c-Jun/c-Fos, and JunD, but not JunB, in binding with AP-1 sites of the PAI-1 promoter upon PlGF induction. Consistent with these findings, levels of PAI-1 were low in PlGF knock-out mice and sickle-PlGF knock-out mice; overexpression of PlGF in normal mice increased circulating PAI-1. In conclusion, we identify a novel mechanism of PAI-1 elevation in SCD.

Hypoxia-mediated expression of 5-lipoxygenase-activating protein involves HIF-1alpha and NF-kappaB and microRNAs 135a and 199a-5p.

Hypoxia occurs in a number of pathological states, such as pulmonary, hematological, and cardiovascular disorders. In this study, we examined the molecular mechanism by which hypoxia contributes to increased leukotriene formation. Our studies showed hypoxia augmented the expression of 5-lipoxygenase activating protein (FLAP), a key enzyme in leukotriene formation, in both human pulmonary microvascular endothelial cells and a transformed human brain endothelial cell line. Hypoxia-induced FLAP mRNA expression involved activation of NADPH-oxidase, PI-3 kinase, mitogen-activated protein kinase, NF-kappaB, and hypoxia-inducible factor (HIF)-1alpha. Hypoxia-induced FLAP promoter activity was attenuated on mutation of hypoxia-response elements (HREs) and NF-kappaB binding motif in the FLAP promoter. Hypoxia also augmented binding of HIF-1alpha to HREs in FLAP promoter as demonstrated by EMSA with nuclear extracts. Furthermore, chromain immunoprecipitation analysis showed HIF-1alpha bound to HREs in native chromatin obtained from hypoxia-treated cells. Next, we examined the role of HIF-1alpha regulated microRNAs on FLAP expression. Our studies showed decreased expression of miR-135a and miR-199a-5p in response to hypoxia. However, overexpression of anti-miR-135a and anti-miR-199a-5p oligonucleotides led to a several fold increased FLAP mRNA and protein expression. These studies demonstrate for the first time that hypoxia-mediated FLAP expression is regulated by HREs and NF-kappaB site in its promoter, and negatively regulated by miR-135a and miR-199a-5p, which target the 3'-UTR of FLAP mRNA. An understanding of these regulatory pathways provides new avenues to ameliorate leukotriene formation and hence reactive airway disease, and inflammation in individuals who have sickle cell disease.

High levels of placenta growth factor in sickle cell disease promote pulmonary hypertension.

Pulmonary hypertension is associated with reduced nitric oxide bioavailability and early mortality in sickle cell disease (SCD). We previously demonstrated that placenta growth factor (PlGF), an angiogenic factor produced by erythroid cells, induces hypoxia-independent expression of the pulmonary vasoconstrictor endothelin-1 in pulmonary endothelial cells. Using a lentivirus vector, we simulated erythroid expression of PlGF in normal mice up to the levels seen in sickle mice. Consequently, endothelin-1 production increased, right ventricle pressures increased, and right ventricle hypertrophy and pulmonary changes occurred in the mice within 8 weeks. These findings were corroborated in 123 patients with SCD, in whom plasma PlGF levels were significantly associated with anemia, endothelin-1, and tricuspid regurgitant velocity; the latter is reflective of peak pulmonary artery pressure. These results illuminate a novel mechanistic pathway linking hemolysis and erythroid hyperplasia to increased PlGF, endothelin-1, and pulmonary hypertension in SCD, and suggest that strategies that block PlGF signaling may be therapeutically beneficial.

Ethanol augments RANTES/CCL5 expression in rat liver sinusoidal endothelial cells and human endothelial cells via activation of NF-kappa B, HIF-1 alpha, and AP-1.

Chronic alcohol consumption leads to liver inflammation and cirrhosis. Alcoholic liver disease patients have increased levels of hepatic RANTES/CCL5. However, less is known about the molecular mechanisms for ethanol-induced RANTES up-regulation. In this study, we observed that liver sinusoidal endothelial cells derived from ethanol-fed rats (E-rLSECs) showed severalfold increases in RANTES and hypoxia-inducible factor 1alpha (HIF-1alpha) mRNAs compared with control rLSECs (C-rLSECs). Similar effects were seen in acute ethanol treatment of isolated rLSECs and human dermal microvascular endothelial cells. Ethanol-induced RANTES mRNA expression required ethanol metabolism, p38 MAPK, HIF-1alpha, and JNK-2, but not JNK-1. EMSA experiments showed increased HIF-1alpha binding to wild-type hypoxia response elements (HREs; -31 to -9 bp) within the RANTES promoter in response to ethanol. RANTES promoter analysis showed that cis elements proximal to the transcription start site, HRE-1 (nt -22 to -19), HRE-2 (nt -32 to -29), and AP-1 (nt -250 to -244) were required for ethanol-mediated RANTES expression. These results were corroborated by chromatin immunoprecipitation assays showing augmented HIF-1alpha binding to HRE-1. Additionally, promoter analysis revealed c-Jun, c-Jun/c-Fos, and JunD, but not JunB, bound to the AP-1 site of the RANTES promoter. Ethanol-mediated activation of NF-kappaB led to HIF-1alpha activation and concomitant RANTES expression. Plasma of ethanol-fed c-Jun(flox/flox)-Mx-1-Cre mice showed attenuated levels of RANTES compared with ethanol-fed control mice, supporting the role of c-Jun in ethanol-induced RANTES expression. Our studies showed that ethanol-mediated RANTES/CCL5 expression occurs via HIF-1alpha activation independently of hypoxia. The identification of HIF-1alpha and AP-1 in ethanol-induced RANTES expression provides new strategies to ameliorate ethanol-induced inflammatory responses.

Ethanol-induced expression of ET-1 and ET-BR in liver sinusoidal endothelial cells and human endothelial cells involves hypoxia-inducible factor-1alpha and microrNA-199.

Chronic alcohol consumption leads to inflammation and cirrhosis of the liver. In this study, we observed that liver sinusoidal endothelial cells (LSEC) derived from ethanol-fed rats showed several fold increases in the mRNA expression of endothelin-1 (ET-1), hypoxia-inducible factor-1alpha (HIF-1alpha), and inflammatory cytochemokines compared with control rat LSEC. We also observed the same results in acute ethanol-treated LSEC from control rats and human dermal microvascular endothelial cells. Ethanol-mediated ET-1 expression involved NADPH oxidase and HIF-1alpha activation. Furthermore, ethanol increased the expression of the ET-1 cognate receptor ET-BR in Kupffer cells and THP-1 monocytic cells, which also involved HIF-1alpha activation. Promoter analysis and chromatin immunoprecipitation showed that hypoxia response element sites in the proximal promoter of ET-1 and ET-BR were required for the binding of HIF-1alpha to up-regulate their expression. We showed that microRNAs, miR-199 among several microRNAs, attenuated HIF-1alpha and ET-1 expression, while anti-miR-199 reversed the effects, suggesting that ethanol-induced miR-199 down-regulation may contribute to augmented HIF-1alpha and ET-1 expression. Our studies, for the first time to our knowledge, show that ethanol-mediated ET-1 and ET-BR expression involve HIF-1alpha, independent of hypoxia. Additionally, ethanol-induced ET-1 expression in rat LSEC is regulated by miR-199, while in human endothelial cells, ET-1 expression is regulated by miR-199 and miR-155, indicating that these microRNAs may function as novel negative regulators to control ET-1 transcription and, thus, homeostatic levels of ET-1 to maintain microcirculatory tone.

Phosphate-buffered saline-based nucleofection of primary endothelial cells.

Although various nonviral transfection methods are available, cell toxicity, low transfection efficiency, and high cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that uses a combination of novel electroporation condition and specific buffer components that stabilize the cells in the electrical field. Despite superior transfection efficiency and cell viability, high cost of the technology has discouraged cardiovascular researchers from liberally adopting this new technology. Here we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular, and lymphatic endothelial cells were performed using PBS. Compared with the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and nonprimary cells. Moreover, PBS-mediated nucleofection of small interfering RNA (siRNA) showed more than 90% knockdown of the expression of target genes in primary endothelial cells. We demonstrate that PBS can be an unprecedented economical alternative to the high-cost buffers or nucleofection of various primary and nonprimary cells.