To kill invading bacteria, neutrophils must interpret spatial cues, migrate, and reach target sites. Although initiation of chemotactic migration has been extensively studied, little is known about its termination. Here we report that two mitogen-activated protein kinases played opposing roles in neutrophil trafficking. The extracellular signal-regulated kinase (Erk) potentiated G protein-coupled receptor kinase GRK2 activity and inhibited neutrophil migration, whereas p38 MAPK acted as a non-canonical GRK that phosphorylated the formyl peptide receptor FPR1 and facilitated neutrophil migration by blocking GRK2 function. Therefore, the dynamic balance between Erk and p38 MAPK controls neutrophil “stop” and “go” behaviors, ensuring neutrophils precisely reach their final destination as the first line of host-defense.

The SphK1/S1P axis is a novel pathway in PAH that promotes PASMC proliferation, a major contributor to pulmonary vascular remodeling. Our results suggest that this pathway is a potential therapeutic target in PAH.

Pulmonary vascular remodeling characterized by concentric wall thickening and intraluminal obliteration is a major contributor to the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Here we report that increased hypoxia-inducible factor 2α (HIF-2α) in lung vascular endothelial cells (LVECs) under normoxic conditions is involved in the development of pulmonary hypertension (PH) by inducing endothelial-to-mesenchymal transition (EndMT), which subsequently results in vascular remodeling and occlusive lesions. We observed significant EndMT and markedly increased expression of SNAI, an inducer of EndMT, in LVECs from patients with IPAH and animals with experimental PH compared with normal controls. LVECs isolated from IPAH patients had a higher level of HIF-2α than that from normal subjects, whereas HIF-1α was upregulated in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. The increased HIF-2α level, due to downregulated prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase that promotes HIF-2α degradation, was involved in enhanced EndMT and upregulated SNAI1/2 in LVECs from patients with IPAH. Moreover, knockdown of HIF-2α (but not HIF-1α) with siRNA decreases both SNAI1 and SNAI2 expression in IPAH-LVECs. Mice with endothelial cell (EC)-specific knockout (KO) of the PHD2 gene, egln1 (egln1), developed severe PH under normoxic conditions, whereas Snai1/2 and EndMT were increased in LVECs of egln1 mice. EC-specific KO of the HIF-2α gene, hif2a, prevented mice from developing hypoxia-induced PH, whereas EC-specific deletion of the HIF-1α gene, hif1a, or smooth muscle cell (SMC)-specific deletion of hif2a, negligibly affected the development of PH. Also, exposure to hypoxia for 48-72 h increased protein level of HIF-1α in normal human PASMCs and HIF-2α in normal human LVECs. These data indicate that increased HIF-2α in LVECs plays a pathogenic role in the development of severe PH by upregulating SNAI1/2, inducing EndMT, and causing obliterative pulmonary vascular lesions and vascular remodeling.

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Summary Background Rare genetic variants cause pulmonary arterial hypertension, but the contribution of common genetic variation to disease risk and natural history is poorly characterised. We tested for genome-wide association for pulmonary arterial hypertension in large international cohorts and assessed the contribution of associated regions to outcomes. Methods We did two separate genome-wide association studies (GWAS) and a meta-analysis of pulmonary arterial hypertension. These GWAS used data from four international case-control studies across 11 744 individuals with European ancestry (including 2085 patients). One GWAS used genotypes from 5895 whole-genome sequences and the other GWAS used genotyping array data from an additional 5849 individuals. Cross-validation of loci reaching genome-wide significance was sought by meta-analysis. Conditional analysis corrected for the most significant variants at each locus was used to resolve signals for multiple associations. We functionally annotated associated variants and tested associations with duration of survival. All-cause mortality was the primary endpoint in survival analyses. Findings A locus near SOX17 (rs10103692, odds ratio 1·80 [95% CI 1·55–2·08], p=5·13 × 10 –15 ) and a second locus in HLA-DPA1 and HLA-DPB1 (collectively referred to as HLA-DPA1/DPB1 here; rs2856830, 1·56 [1·42–1·71], p=7·65 × 10 –20 ) within the class II MHC region were associated with pulmonary arterial hypertension. The SOX17 locus had two independent signals associated with pulmonary arterial hypertension (rs13266183, 1·36 [1·25–1·48], p=1·69 × 10 –12 ; and rs10103692). Functional and epigenomic data indicate that the risk variants near SOX17 alter gene regulation via an enhancer active in endothelial cells. Pulmonary arterial hypertension risk variants determined haplotype-specific enhancer activity, and CRISPR-mediated inhibition of the enhancer reduced SOX17 expression. The HLA-DPA1/DPB1 rs2856830 genotype was strongly associated with survival. Median survival from diagnosis in patients with pulmonary arterial hypertension with the C/C homozygous genotype was double (13·50 years [95% CI 12·07 to >13·50]) that of those with the T/T genotype (6·97 years [6·02–8·05]), despite similar baseline disease severity. Interpretation This is the first study to report that common genetic variation at loci in an enhancer near SOX17 and in HLA-DPA1/DPB1 is associated with pulmonary arterial hypertension. Impairment of SOX17 function might be more common in pulmonary arterial hypertension than suggested by rare mutations in ...

In neutrophils, the phosphoinositide 3-kinase/Akt signaling cascade is involved in migration, degranulation, and O 2 ؊ production. However, it is unclear whether the Akt kinase isoforms have distinct functions in neutrophil activation. Here we report functional differences between the 2 major Akt isoforms in neutrophil activation on the basis of studies in which we used individual Akt1 and Akt2 knockout mice. Akt2 ؊/؊ neutrophils exhibited decreased cell migration, granule enzyme release, and O 2 ؊ production compared with wild-type and Akt1 ؊/؊ neutrophils. Surprisingly, Akt2 deficiency and pharmacologic inhibition of Akt also abrogated phorbol ester-induced O 2 ؊ production, which was unaffected by treatment with the phosphoinositide 3-kinase inhibitor LY294002. The decreased O 2 ؊ production in Akt2 ؊/؊ neutrophils was accompanied by reduced p47 phox phosphorylation and its membrane translocation, suggesting that Akt2 is important for the assembly of phagocyte nicotinamide adenine dinucleotide phosphate oxidase.In wild-type neutrophils, Akt2 but not Akt1 translocated to plasma membrane upon chemoattractant stimulation and to the leading edge in polarized neutrophils. In the absence of Akt2, chemoattractantinduced Akt protein phosphorylation was significantly reduced. These results demonstrate a predominant role of Akt2 in regulating neutrophil functions and provide evidence for differential activation of the 2 Akt isoforms in neutrophils. (Blood. 2010;115(21):4237-4246) IntroductionAkt, also known as protein kinase B (PKB), constitutes a family of serine/threonine kinases with a characteristic pleckstrin homology (PH) domain in the N-terminus followed by a catalytic domain and a C-terminal regulatory domain. Akt has 3 isoforms, Akt1 (PKB␣), Akt2 (PKB␤), and Akt3 (PKB␥). Akt1 was initially identified through homologous DNA cloning in 1991 and was found as a cellular homologue of the viral oncogene v-akt from the acutely transforming retrovirus Akt8. 1 Akt2 was identified as an overexpressed protein in ovarian carcinoma cell lines and primary ovarian tumors. 2 Akt3 initially was cloned from the rat brain and testis. 3 These Akt isoforms share a high degree of homology, especially in the catalytic domains (87%-90% identical amino acids). Walker et al, 4 in an vitro study, found no discernable difference between Akt1, Akt2, and Akt3 in their ability to recognize and phosphorylate peptide substrate. In other studies, 5-8 the individual Akt isoforms were found to distribute differently and have different functions despite the high level of homology between these isoforms. Akt1 is expressed in most tissues and promotes cell survival by inhibiting apoptosis. Akt1 also induces protein synthesis and is crucial to growth and development as shown in studies that used Akt1 Ϫ/Ϫ mice. [9][10][11] Akt2 is expressed mainly in insulin-responsive organs, including liver, skeletal muscle, and adipose tissue. Consistent with its primary function in insulin signaling, the Akt2 Ϫ/Ϫ mice display a type 2 diabetic phenotype. [12][13]...

Rationale: Recent studies suggest that microRNAs (miRNAs) play important roles in regulation of pulmonary artery smooth muscle cell (PASMC) phenotype and are implicated in pulmonary arterial hypertension (PAH). However, the underlying molecular mechanisms remain elusive.Objectives: This study aims to understand the mechanisms regulating PASMC proliferation and differentiation by microRNA-17z92 (miR-17z92) and to elucidate its implication in PAH. Methods:We generated smooth muscle cell (SMC)-specific miR17z92 and PDZ and LIM domain 5 (PDLIM5) knockout mice and overexpressed miR-17z92 and PDLIM5 by injection of miR-17z92 mimics or PDLIM5-V5-His plasmids and measured their responses to hypoxia. We used miR-17z92 mimics, inhibitors, overexpression vectors, small interfering RNAs against PDLIM5, Smad, and transforming growth factor (TGF)-b to determine the role of miR-17z92 and its downstream targets in PASMC proliferation and differentiation.Measurements and Main Results: We found that human PASMC (HPASMC) from patients with PAH expressed decreased levels of the miR-17z92 cluster, TGF-b, and SMC markers. Overexpression of miR-17z92 increased and restored the expression of TGF-b 3 , Smad3, and SMC markers in HPASMC of normal subjects and patients with idiopathic PAH, respectively. Knockdown of Smad3 but not Smad2 prevented miR-17z92-induced expression of SMC markers. SMC-specific knockout of miR-17z92 attenuated hypoxiainduced pulmonary hypertension (PH) in mice, whereas reconstitution of miR-17z92 restored hypoxia-induced PH in these mice. We also found that PDLIM5 is a direct target of miR-17/20a, and hypertensive HPASMC and mouse PASMC expressed elevated PDLIM5 levels. Suppression of PDLIM5 increased expression of SMC markers and enhanced TGF-b/Smad2/3 activity in vitro and enhanced hypoxia-induced PH in vivo, whereas overexpression of PDLIM5 attenuated hypoxia-induced PH. Conclusions:We provided the first evidence that miR-17z92 inhibits PDLIM5 to induce the TGF-b 3 /SMAD3 pathway, contributing to the pathogenesis of PAH.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological response that optimizes the ventilation/perfusion ratio. Chronic hypoxia causes vascular remodeling, which is central to the pathogenesis of hypoxia-induced pulmonary hypertension (HPH). We have previously shown that Notch3 is up-regulated in HPH and that activation of Notch signaling enhances store-operated Ca 21 entry (SOCE), an important mechanism that contributes to pulmonary arterial smooth muscle cell (PASMC) proliferation and contraction. Here, we investigate the role of Notch signaling in HPV and hypoxia-induced enhancement of SOCE. We examined SOCE in human PASMCs exposed to hypoxia and pulmonary arterial pressure in mice using the isolated perfused/ventilated lung method. Wildtype and canonical transient receptor potential (TRPC) 6 2/2 mice were exposed to chronic hypoxia to induce HPH. Inhibition of Notch signaling with a g-secretase inhibitor attenuates hypoxia-enhanced SOCE in PASMCs and hypoxia-induced increase in pulmonary arterial pressure. Our results demonstrate that hypoxia activates Notch signaling and up-regulates TRPC6 channels. Additionally, treatment with a Notch ligand can mimic hypoxic responses. Finally, inhibition of TRPC6, either pharmacologically or genetically, attenuates HPV, hypoxia-enhanced SOCE, and the development of HPH. These results demonstrate that hypoxia-induced activation of Notch signaling mediates HPV and the development of HPH via functional activation and up-regulation of TRPC6 channels. Understanding the molecular mechanisms that regulate cytosolic free Ca 21 concentration and PASMC proliferation is critical to elucidation of the pathogenesis of HPH. Targeting Notch regulation of TRPC6 will be beneficial in the development of novel therapies for pulmonary hypertension associated with hypoxia.