Tissue hypoxemia is common in several pathological diseases, including vaso-occlusion in sickle cell disease and myocardial infarction. One finds increased presence of leukocytes during lung injury and at sites of inflammation in vascular endothelium. In this study, we used human pulmonary microvascular endothelial cells and human dermal microvascular endothelial immortalized cell line to delineate the cellular signaling mechanism of hypoxia- and CoCl2 (a mimetic of hypoxia)-induced IL-8 expression, and the latter’s role in chemotaxis of polmorphonuclear neutrophils. We show that hypoxia- and CoCl2-induced IL-8 mRNA and protein expression involved activation of PI3K/Akt and p38 MAPK, but not MEK kinase. Analysis of some transcription factors associated with IL-8 promoter revealed that hypoxia and CoCl2 increased DNA-binding activity of hypoxia-inducible factor-1α (HIF-1α), NF-κB, and AP-1. In addition, we show that hypoxia- and CoCl2-induced IL-8 expression requires activation of HIF as demonstrated by the following: 1) EMSA; 2) transfection studies with IL-8 promoter reporter constructs with mutation in HIF-1α binding site; 3) attenuation of IL-8 expression by both HIF-1α small interfering RNA and R59949; 4) augmentation of IL-8 expression by either transfection with HIF-prolyl hydroxylase-2 small interfering RNA or overexpression of HIF-1α; and 5) chromatin immunoprecipitation analysis. Moreover, conditioned medium from hypoxia-treated endothelial cells augmented chemotaxis of neutrophils, due to release of IL-8. These data indicate that hypoxia-induced signaling in vascular endothelium for transcriptional activation of IL-8 involves PI3K/Akt, p38 MAPK, and HIF-1α. Pharmacological agents, which inhibit HIF-1α, may possibly ameliorate inflammation associated with hypoxia in pathological diseases.
Pulmonary hypertension (PHT) develops in sickle cell disease (SCD) and is associated with high mortality. We previously showed that erythroid cells produce placenta growth factor (PlGF), which activates monocytes to induce proinflammatory cytochemokines, contributing to the baseline inflammation and severity in SCD. In this study, we observed that PlGF increased expression of endothelin-1 (ET-1) and endothelin-B receptor (ET-BR) from human pulmonary microvascular endothelial cells (HPMVECs) and monocytes, respectively. PlGF-mediated ET-1 and ET-BR expression occurred via activation of PI-3 kinase, reactive oxygen species and hypoxia inducible factor-1␣ (HIF-1␣). PlGF increased binding of HIF-1␣ to the ET-1 and ET-BR promoters; this effect was abrogated with mutation of hypoxia response elements in the promoter regions and HIF-1␣ siRNA and confirmed by chromatin immunoprecipitation analysis. Furthermore, PlGF-mediated ET-1 release from HPMVECs and ET-BR expression in monocytes creates a PlGF-ET-1-ET-BR loop, leading to increased expression of MCP-1 and IL-8. Our studies show that PlGF-induced expression of the potent vasoconstrictor ET-1 and its cognate ET-BR receptor occur via activation of HIF-1␣, independent of hypoxia. PlGF levels are intrinsically elevated from the increased red cell turnover in SCD and in other chronic anemia (eg, thalassemia) and may contribute to inflammation and PHT seen in these diseases. (Blood. 2008;112:856-865) IntroductionThe clinical manifestations of sickle cell disease (SCD) include chronic hemolytic anemia, frequent infections, and intermittent episodes of vascular occlusion. [1][2][3][4][5][6] Pulmonary disease, both acute and chronic, is the second most common cause of hospitalization and a leading cause of both morbidity and mortality in adults with SCD. 1,[7][8][9] The most frequent form of acute pulmonary disease is the acute chest syndrome (ACS), which occurs in 15% to 40% of patients with SCD. 10 Pulmonary hypertension (PHT) occurs in both adults and children with SCD: it develops with increasing age and portends an extremely poor prognosis. PHT is a significant risk factor for early mortality in SCD. 9,[11][12][13] Studies have shown that there is a clinical syndrome of hemolysis-associated PHT in SCD 9 that results from global impairment in nitric oxide (NO) bioavailability from its quenching by free heme. 14 It is known that vascular tone is also modulated by vasoconstrictors such as endothelin-1 (ET-1). Studies have shown increased plasma levels of ET-1 in patients with SCD and ACS. 15,16 Tissue hypoxemia due to microvascular occlusion and chronic mild-moderate desaturations in SCD may contribute to increased levels of ET-1, which is released from endothelial cells in response to hypoxia. 17 Increased levels of ET-1, if sustained as a result of the underlying SCD pathophysiology, can contribute to the development of PHT.SCD is also characterized by presence of a chronic inflammatory state manifested by leukocytosis and monocytosis and increased circulating levels of p...
Background: Elevated plasma levels of PlGF are associated with increased endothelin-1 and pulmonary hypertension (PH) in SCD. Results: miR-199a2, which targets HIF-1␣ mRNA, located in host gene DNM3os is co-transcriptionally regulated by PPAR␣. Conclusion: PPAR␣ agonist induction of miR-199a2 reduced ET-1 levels. Significance: PPAR␣ agonist reduction of ET-1 levels via induced miR-199a2 provides an alternative strategy to ameliorate PH.
IntroductionInflammation is increasingly recognized as central to the pathophysiology of sickle cell disease (SCD) and is manifest as leukocytosis, elevated levels of inflammatory cytokines, and activation of neutrophils, monocytes, and endothelial cells. [1][2][3][4] It is present at steady state and is strongly associated with acute painful events, acute chest, and early mortality. 5,6 Current evidence strongly suggests that inflammation contributes to the endothelial cell dysfunction, potentiates vasoocclusion, and may also give rise to the airway hyperreactivity (AH) that often accompanies SCD. [7][8][9][10] Also intriguing is the spectrum of lung disease seen in this patient population, which spans from an increased incidence of AH and obstructive lung disease in children, [11][12][13] to restrictive lung disease and pulmonary vascular remodeling, which is associated with pulmonary hypertension in adults. [14][15][16][17][18] Leukotrienes (LT) mediate both inflammation and AH. 19-22 5-Lipoxygenase (5-LO) and its activating partner, 5-lipoxygenase activating protein (FLAP), catalyze the production of LT from arachidonic acid (AA) by generating 5-hydroperoxyeicostatraenoic acid (5-HPETE) and leukotriene A 4 (LTA 4 ). LTA 4 is the pivotal intermediate from which other LTs (ie, LTB 4 and cysteinyl LT [CysLT], LTC 4 , LTD 4 , and LTE 4 ) are formed. 20 LTB 4 is one of the most potent chemoattractant for neutrophils, eosinophils, and mediator of inflammation. CysLT, on the other hand, are potent bronchoconstrictors that play an important role in edema, inflammation, and mucus secretion in asthma and were previously termed "slow releasing substances." 23 LT play an important role in the pathogenesis of inflammatory disorders, specifically asthma, rheumatoid arthritis, and inflammatory bowel disease. [19][20][21] Studies by Bigby and coworkers 24,25 have shown that both tumor necrosis factor-␣ (TNF-␣) and lipopolysaccharide (LPS) induce the expression of FLAP in THP-1 cells. These studies showed the importance of nuclear factor-B (NF-B) and CCAAT/enhancer binding protein (C/EBP) transcription factors in the LPS-mediated FLAP expression. 24 LTB 4 levels are higher in SCD patients at steady state, which are further increased in vasoocclusive pain crises (VOC) and acute chest syndrome (ACS). 26 Very recently, increased LTE 4 has been observed in patients with SCD, which is associated with a higher incidence of pain. 27 However, less is understood about how LTs are increased in SCD at the molecular level.Placenta growth factor (PlGF) is an angiogenic growth factor with similar effects on endothelium as vascular endothelial growth factor (VEGF) and is primarily expressed by placental trophoblasts. [28][29][30] More recently, we and others show that erythroid cells, but not other hematopoietic cells, produce PlGF, and its expression is high in SCD and thalassemia. 31,32 VEGFR1 is its cognate receptor and is expressed on endothelial cells, alveolar epithelial cells, mast cells, and monocytes. We have previously shown that...
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-κB, and hypoxia-inducible factor (HIF)-1α. Hypoxia-induced FLAP promoter activity was attenuated on mutation of hypoxia-response elements (HREs) and NF-κB binding motif in the FLAP promoter. Hypoxia also augmented binding of HIF-1α to HREs in FLAP promoter as demonstrated by EMSA with nuclear extracts. Furthermore, chromain immunoprecipitation analysis showed HIF-1α bound to HREs in native chromatin obtained from hypoxia-treated cells. Next, we examined the role of HIF-1α 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-κB 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.
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