Justin R. Sysol

The sphingosine kinase 1/sphingosine-1-phosphate pathway in pulmonary arterial hypertension

RATIONALE Sphingosine kinases (SphKs) 1 and 2 regulate the synthesis of the bioactive sphingolipid sphingosine-1-phosphate (S1P), an important lipid mediator that promotes cell proliferation, migration, and angiogenesis. OBJECTIVES We aimed to examine whether SphKs and their product, S1P, play a role in the development of pulmonary arterial hypertension (PAH). METHODS SphK1(-/-), SphK2(-/-), and S1P lyase heterozygous (Sgpl1(+/-)) mice, a pharmacologic SphK inhibitor (SKI2), and a S1P receptor 2 (S1PR2) antagonist (JTE013) were used in rodent models of hypoxia-mediated pulmonary hypertension (HPH). S1P levels in lung tissues from patients with PAH and pulmonary arteries (PAs) from rodent models of HPH were measured. MEASUREMENTS AND MAIN RESULTS mRNA and protein levels of SphK1, but not SphK2, were significantly increased in the lungs and isolated PA smooth muscle cells (PASMCs) from patients with PAH, and in lungs of experimental rodent models of HPH. S1P levels were increased in lungs of patients with PAH and PAs from rodent models of HPH. Unlike SphK2(-/-) mice, SphK1(-/-) mice were protected against HPH, whereas Sgpl1(+/-) mice were more susceptible to HPH. Pharmacologic SphK1 and S1PR2 inhibition prevented the development of HPH in rodent models of HPH. Overexpression of SphK1 and stimulation with S1P potentially via ligation of S1PR2 promoted PASMC proliferation in vitro, whereas SphK1 deficiency inhibited PASMC proliferation. CONCLUSIONS 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.

Deficiency of Akt1, but not Akt2, attenuates the development of pulmonary hypertension

Pulmonary vascular remodeling, mainly attributable to enhanced pulmonary arterial smooth muscle cell proliferation and migration, is a major cause for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with pulmonary hypertension. The signaling cascade through Akt, comprised of three isoforms (Akt1-3) with distinct but overlapping functions, is involved in regulating cell proliferation and migration. This study aims to investigate whether the Akt/mammalian target of rapamycin (mTOR) pathway, and particularly which Akt isoform, contributes to the development and progression of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Compared with the wild-type littermates, Akt1(-/-) mice were protected against the development and progression of chronic HPH, whereas Akt2(-/-) mice did not demonstrate any significant protection against the development of HPH. Furthermore, pulmonary vascular remodeling was significantly attenuated in the Akt1(-/-) mice, with no significant effect noted in the Akt2(-/-) mice after chronic exposure to normobaric hypoxia (10% O2). Overexpression of the upstream repressor of Akt signaling, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and conditional and inducible knockout of mTOR in smooth muscle cells were also shown to attenuate the rise in right ventricular systolic pressure and the development of right ventricular hypertrophy. In conclusion, Akt isoforms appear to have a unique function within the pulmonary vasculature, with the Akt1 isoform having a dominant role in pulmonary vascular remodeling associated with HPH. The PTEN/Akt1/mTOR signaling pathway will continue to be a critical area of study in the pathogenesis of pulmonary hypertension, and specific Akt isoforms may help specify therapeutic targets for the treatment of pulmonary hypertension.

Nicotinamide Phosphoribosyltransferase Promotes Pulmonary Vascular Remodeling and is a Therapeutic Target in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension is a severe and progressive disease, a hallmark of which is pulmonary vascular remodeling. Nicotinamide phosphoribosyltransferase (NAMPT) is a cytozyme that regulates intracellular nicotinamide adenine dinucleotide levels and cellular redox state, regulates histone deacetylases, promotes cell proliferation, and inhibits apoptosis. We hypothesized that NAMPT promotes pulmonary vascular remodeling and that inhibition of NAMPT could attenuate pulmonary hypertension. Methods: Plasma, mRNA, and protein levels of NAMPT were measured in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension and in the lungs of rodent models of pulmonary hypertension. Nampt+/− mice were exposed to 10% hypoxia and room air for 4 weeks, and the preventive and therapeutic effects of NAMPT inhibition were tested in the monocrotaline and Sugen hypoxia models of pulmonary hypertension. The effects of NAMPT activity on proliferation, migration, apoptosis, and calcium signaling were tested in human pulmonary artery smooth muscle cells. Results: Plasma and mRNA and protein levels of NAMPT were increased in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension, as well as in lungs of rodent models of pulmonary hypertension. Nampt+/− mice were protected from hypoxia-mediated pulmonary hypertension. NAMPT activity promoted human pulmonary artery smooth muscle cell proliferation via a paracrine effect. In addition, recombinant NAMPT stimulated human pulmonary artery smooth muscle cell proliferation via enhancement of store-operated calcium entry by enhancing expression of Orai2 and STIM2. Last, inhibition of NAMPT activity attenuated monocrotaline and Sugen hypoxia–induced pulmonary hypertension in rats. Conclusions: Our data provide evidence that NAMPT plays a role in pulmonary vascular remodeling and that its inhibition could be a potential therapeutic target for pulmonary arterial hypertension.

Hemin Causes Lung Microvascular Endothelial Barrier Dysfunction by Necroptotic Cell Death

&NA; Hemin, the oxidized prosthetic moiety of hemoglobin, has been implicated in the pathogenesis of acute chest syndrome in patients with sickle cell disease by virtue of its endothelial‐activating properties. In this study, we examined whether hemin can cause lung microvascular endothelial barrier dysfunction. By assessing transendothelial resistance using electrical cell impedance sensing, and by directly measuring trans‐monolayer fluorescein isothiocyanate‐dextran flux, we found that hemin does cause endothelial barrier dysfunction in a concentration‐dependent manner. Pretreatment with either a Toll‐like receptor 4 inhibitor, TAK‐242, or an antioxidant, N‐acetylcysteine, abrogated this effect. Increased monolayer permeability was found to be associated with programmed cell death by necroptosis, as evidenced by Trypan blue staining, terminal deoxynucleotidyl transferase dUTP nick‐end labeling assay, Western blotting for activated forms of key effectors of cell death pathways, and studies utilizing specific inhibitors of necroptosis and apoptosis. Further studies examining the role of endothelial cell necroptosis in promoting noncardiogenic pulmonary edema during acute chest syndrome are warranted and may open a new avenue of potential treatments for this devastating disease.

Genome-Wide Analysis Identifies IL-18 and FUCA2 as Novel Genes Associated with Diastolic Function in African Americans with Sickle Cell Disease

Background Diastolic dysfunction is common in sickle cell disease (SCD), and is associated with an increased risk of mortality. However, the molecular pathogenesis underlying this development is poorly understood. The aim of this study was to identify a gene expression profile that is associated with diastolic function in SCD, potentially elucidating molecular mechanisms behind diastolic dysfunction development. Methods Diastolic function was measured via echocardiography in 65 patients with SCD from two independent study populations. Gene expression microarray data was compared with diastolic function in both study cohorts. Candidate genes that associated in both analyses were tested for validation in a murine SCD model. Lastly, genotyping array data from the replication cohort was used to derive cis-expression quantitative trait loci (cis-eQTLs) and genetic associations within the candidate gene regions. Results Transcriptome data from both patient cohorts implicated 7 genes associated with diastolic function, and mouse SCD myocardial expression validated 3 of these genes. Genetic associations and eQTLs were detected in 2 of the 3 genes, FUCA2 and IL18. Conclusions FUCA2 and IL18 are associated with diastolic function in SCD patients, and may be involved in the pathogenesis of the disease. Genetic polymorphisms within the FUCA2 and IL18 gene regions are also associated with diastolic function in SCD, likely by affecting expression levels of the genes.

Loss of lung WWOX expression causes neutrophilic inflammation

The tumor suppressor WW domain-containing oxidoreductase (WWOX) exhibits regulatory interactions with an array of transcription factors and signaling molecules that are positioned at the well-known crossroads between inflammation and cancer. WWOX is also subject to downregulation by genotoxic environmental exposures, making it of potential interest to the study of lung pathobiology. Knockdown of lung WWOX expression in mice was observed to cause neutrophil influx and was accompanied by a corresponding vascular leak and inflammatory cytokine production. In cultured human alveolar epithelial cells, loss of WWOX expression resulted in increased c-Jun- and IL-8-dependent neutrophil chemotaxis toward cell monolayers. WWOX was observed to directly interact with c-Jun in these cells, and its absence resulted in increased nuclear translocation of c-Jun. Finally, inhibition of the c-Jun-activating kinase JNK abrogated the lung neutrophil influx observed during WWOX knockdown in mice. Altogether, these observations represent a novel mechanism of pulmonary neutrophil influx that is highly relevant to the pathobiology and potential treatment of a number of different lung inflammatory conditions.

Progressive glomerular and tubular damage in sickle cell trait and sickle cell anemia mouse models

&NA; Homozygosity for the hemoglobin (Hb) S mutation (HbSS, sickle cell anemia) results in hemoglobin polymerization under hypoxic conditions leading to vaso‐occlusion and hemolysis. Sickle cell anemia affects 1:500 African Americans and is a strong risk factor for kidney disease, although the mechanisms are not well understood. Heterozygous inheritance (HbAS; sickle cell trait) affects 1:10 African Americans and is associated with an increased risk for kidney disease in some reports. Using transgenic sickle mice, we investigated the histopathologic, ultrastructural, and gene expression differences with the HbS mutation. Consistent with progressive glomerular damage, we observed progressively greater urine protein concentrations (P = 0.03), glomerular hypertrophy (P = 0.002), and glomerular cellularity (P = 0.01) in HbAA, HbAS, and HbSS mice, respectively. Ultrastructural studies demonstrated progressive podocyte foot process effacement, glomerular basement membrane thickening with reduplication, and tubular villous atrophy with the HbS mutation. Gene expression studies highlighted the differential expression of several genes involved in prostaglandin metabolism (AKR1C18), heme and iron metabolism (HbA‐A2, HMOX1, SCL25A37), electrolyte balance (SLC4A1, AQP6), immunity (RSAD2, C3, UBE2O), fatty acid metabolism (FASN), hypoxia hall‐mark genes (GCK, SDC3, VEGFA, ETS1, CP, BCL2), as well as genes implicated in other forms of kidney disease (PODXL, ELMO1, FRMD3, MYH9, APOA1). Pathway analysis highlighted increased gene enrichment in focal adhesion, extracellular matrix‐receptor interaction, and axon guidance pathways. In summary, using transgenic sickle mice, we observed that inheritance of the HbS mutation is associated with glomerular and tubular damage and identified several candidate genes and pathways for future investigation in sickle cell trait and sickle cell anemia‐related kidney disease.

Sickle Cell Disease and Acute Chest Syndrome: Epidemiology, Diagnosis, Management, Outcomes

Sickle cell disease (SCD) is one of the most common monogenetic diseases worldwide and is attributable to significant morbidity and mortality. Mutations causing abnormal hemoglobin formation in this disease lead to structural abnormalities and cumulative damage to the cellular membrane of sickled erythrocytes. Polymerization and aggregation of these cells within the microvasculature results in severe vaso-occlusive pain crisis, chronic hemolytic anemia, and multiorgan pathology in patients. Pulmonary manifestations of SCD, including the acute chest syndrome (ACS), are a leading cause of hospitalization and mortality. ACS is a severe type of acute lung injury, defined as the development of a new pulmonary infiltrate, involving at least one complete lung segment, that is accompanied by fever, chest pain, tachypnea, wheezing, or cough in a patient with SCD. The etiology of ACS is multifactorial, with the most common mechanisms including infection, fat and bone marrow embolism, and direct microvascular vaso-occlusion. Despite recent advances in our understanding of the pathogenesis and clinical management of ACS in SCD, patient outcomes remain unacceptably poor. This chapter reviews the epidemiology, diagnosis, management, and outcomes of ACS in SCD. Proper screening, a high index of clinical suspicion, and immediate clinical care for this condition are pivotal for improving patient outcomes.

ID: 139: PROGRESSIVE GLOMERULAR DAMAGE IN SICKLE CELL TRAIT AND SICKLE CELL ANEMIA MOUSE MODELS

The hemoglobin S mutation, a glutamic acid to valine substitution in the β-globin chain, results in hemoglobin polymerization under hypoxic conditions and leads to vaso-occlusion and hemolysis. Homozygous inheritance (Hb SS; sickle cell anemia) affects 1 in 500 African Americans and is consistently associated with an increased risk for kidney disease which may be due to cell-free hemoglobin toxicity, ischemic injury, or hyperfiltration-mediated damage to the kidney. Heterozygous inheritance (Hb AS; sickle cell trait) affects 1 in 8 African Americans and has also been associated with an increased risk for kidney disease, although not in all cohorts and the mechanisms are not well understood. We investigated whether inheritance of the Hb S mutation resulted in incremental kidney damage in Hb AS and Hb SS mice compared to Hb AA mice by histology, proteinuria, and candidate gene expression using transgenic sickle mice ≥6 months of age (Townes model, Jackson Laboratory). Values are presented as mean±standard error and analyses are adjusted for age. Using Masson trichrome stained sections of the kidney, progressive patterns of mesangial expansion were observed in age-matched Hb AS and Hb SS mice versus Hb AA mice by renal pathologists blinded to the hemoglobin genotype (figure 1). Hb AS mice had diffuse (>50% of the glomeruli per slide being involved) mesangial expansion while Hb SS mice had diffuse and global (>50% of the individual glomerulus being involved) mesangial expansion. Glomerular perimeters were measured using NanoZoomer Whole Slide Imaging in 26 randomly selected glomeruli from 2 age-matched mice per genotype. Using the upper quartile as the definition for an enlarged glomerulus, the proportion of enlarged glomeruli progressively increased from Hb AA (15%) to Hb AS (31%) to Hb SS mice (58%) (Cochrans test of linear trend, P=0.001) (figure 2). Progressively higher kidney weights were also observed from Hb AA (429±28 mg, n=8) to Hb AS (446±27 mg, n=18) to Hb SS (567±19 mg, n=5) mice (Test for linear trend, P=0.047). We then measured urine protein and urine creatinine concentrations using the Bio-Rad dye method and Jaffé reaction, respectively. Progressively higher urine protein-to-creatinine ratios were observed from Hb AA to Hb AS to Hb SS mice (figure 3) (Test for linear trend, P=0.09). Gene expression of candidate genes (TGFB1, IL6, MMP9, Klotho, HMOX1, and SHROOM3) was determined by rt-PCR from kidneys of age-matched, female Hb AA and Hb AS mice (n=5). Increased expression of Klotho (P=0.09) was observed in Hb AS mice (figure 4). Klotho is a β-glucoronidase that is highly expressed in the kidney and acts as a cofactor that increases the affinity of the FGF23 ligand for the FGF receptor. In conclusion, we observed progressive glomerular injury, determined by mesangial expansion, proportion of enlarged glomeruli, and urine protein concentrations in Hb AS and Hb SS mice compared to Hb AA mice. Klotho was upregulated in Hb AS mice and may play a role in the pathophysiology of kidney damage in Hb AS which will require further investigation. Abstract ID: 139 Figure 1

ID: 72: INHIBITION OF NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (NAMPT) ATTENUATES EXPERIMENTAL PULMONARY HYPERTENSION

Rationale We have previously shown that Nampt, which regulates intracellular NAD levels and cellular redox state, regulates histone deacetylases and inhibits apoptosis, is significantly upregulated in patients with pulmonary arterial hypertension (PAH). The aims of this study were to determine (1) whether Nampt+/− mice are protected from hypoxia-mediated pulmonary hypertension (HPH), (2) whether pharmacological inhibition of Nampt could attenuate monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats. In addition, we hypothesized that Nampt secreted from pulmonary artery endothelial cells (PAECs) or overexpressing Nampt in pulmonary artery smooth muscle cells (PASMCs) may promote PASMC proliferation via upregulation of calcium signaling pathway, which plays a role in cell proliferation and vascular constriction. Methods Nampt+/− mice and their WT siblings (male, 7-wk old) were exposed to a hypoxia chamber with 10% O2 for four weeks. Male Sprague-Dawley rats (n=6 per group) received one dose of MCT (60 mg/kg), IP. They were administrated with FK866 (an inhibitor of Nampt enzymatic activity) (2.5 mg/kg, IP, twice daily for 2wks) two weeks after MCT. Right ventricular systolic pressure (RVSP) was determined with a pressure transducer catheter. The right ventricle: left ventricle+septum (RV/LV+S) ratio was calculated. In a cell culture model, hPASMCs were stimulated with recombinant Nampt (25 mg/ml) for 6 hrs and 48 hrs. [Ca2+]cyt was measured in PASMC loaded with flura-2/AM (4mM) in a fluorescence microscope and cyclepiazonic acid (CPA, a specific Ca2+-ATPase inhibitor) was used to induce store-operated calcium entry (SOCE). In addition, BrdU assays were conducted to examine rNampt or overexpressing Nampt can promote PASMC proliferation or Nampt secreted from PAECs isolated from PAH patients stimulates more PASMC proliferation than from healthy controls. Results Administration of FK866 reversed established PH (RVSP [mm Hg] 19.77±0.80 [control] vs 51.24±4.35 [MCT] vs 34.45±3.49 [MCT+FK866], p<0.05 ) and RVH (0.25±0.0013 vs 0.60±0.019 vs 0.43±0.022, p<0.01). In PASMCs, short (6 hrs) and long (48 hrs) treatment with recombinant PBEF enhanced SOCE which is involved in sustained pulmonary vasoconstriction and PASMC proliferation. rNampt promotes PASMC proliferation in a dose dependent manner. PAECs from PAH patients secreted more Nampt which stimulates more PASMC proliferation compared to healthy controls. Overexpressed Nampt promotes PASMC proliferation. Inhibition of Nampt via FK866 attenuates rNampt-, Nampt overexpressed or PAEC-secreted Nampt – mediated PASMC proliferation. Conclusion Inhibition of Nampt attenuates hypoxia-mediated PH in mice or MCT-induced PH in rats. Nampt may play a role in vascular remodeling via regulation of calcium signaling pathway. These data suggest that Nampt inhibition could be a potential therapeutic target for PH.