Nitric oxide (NO): NO production by endothelial nitric oxide synthase (eNOS; NOS-3) is cytoprotective and regulates smooth muscle tone, leukocyte adhesion, and platelet aggregation. Inducible NOS (iNOS; NOS-2) during sepsis produces large amounts of NO resulting in shock, myocardial depression, tissue injury and apoptosis. This dual nature of NO underlies the paradox that both under and over production are associated with vascular dysfunction. Our work has focused on cGMP-independent, non-canonical NO signaling and inflammatory gene regulation. NO was shown to up-regulate TNFα production (J Immunol 1994; Blood 1997) through a cGMP-independent signaling pathway (J Biol Chem 1997) that utilized NO-responsive Sp1 promoter binding sites (J Biol Chem 1999; J Biol Chem 2003). Dysfunctional eNOS found to upregulate TNFα (J Biol Chem 2000) through the release of reactive oxygen species that activated ERK1/2 (Am J Physiol 2001). NO activation of p38 MAPK stabilized IL-8 mRNA (J Infect Dis 1998) through AU-rich elements in the IL-8 3 UTR (J Leuk Biol 2004). Additional mechanisms involving ERK1/2 and CU-rich elements in other target transcripts demonstrated the diversity of NO effects on transcript stability and translation (Nucleic Acids Research 2006; J Leuk Biol 2008). Sickle cell disease was shown to cause oxidant and inflammatory stress in the vasculature (Blood, 2004). Circulatory stress in sickle cell disease was associated with gene expression changes and arginine metabolism abnormalities (Circulation, 2007). Anti-proliferative effects of NO were linked to p38 MAPK activation and p21 mRNA stabilization with subsequent down-regulation of polo-like kinase 1 through a CDE/CHR proximal promoter site (BMC Genomics 2005; J Biol Chem 2006). Both NO and peroxisome proliferator-activated receptors (PPARs) protect the endothelium and regulate its function. PPARγ was activated by NO through a p38 MAPK dependent signal transduction pathway (FASEB J 2007). In contrast to the pro-inflammatory effects of high output NO, CO was found to block proximal events in NF-κB signal transduction, thereby broadly suppressing inflammation (PLoS One 2009). Nuclear receptors: Nuclear receptors (NRs) regulate cardiovascular homeostasis, metabolism and the immune system. Of 48 human NRs, the glucocorticoid receptor (GR) has been the most extensively studied as a suppressor of inflammation. Glucocorticoid (GC) activation of GR is used to treat a wide range of inflammatory.. In addition to the induction of anti-inflammatory mediators, GR suppresses inflammatory responses by tethering to DNA-bound NF-κB and AP-1 complexes, transcription factor families that broadly control the expression of cytokines, chemokines and adhesion molecules. This trans-repression mechanism has now been described for a growing number of other NRs. PPARγ, MR, AR, and COUP-TF are prominently expressed in human endothelium. In human endothelial cells MR agonists were found to repress NF-κB mediated gene transcription (Keystone Symposia on Nuclear Receptors abstract 2010; American Thoracic Society abstract 2010). In contrast, MR either repressed or further activated AP1 signaling depending on the DNA sequence of specific AP1 binding sites, MR ligand structure, and AP1 family member expression. This investigation demonstrated a novel mechanism for the pro-inflammatory effects of MR (manuscript submitted 2015). Rosiglitazone (RGZ), a PPARγ ligand/agonist, also activates G-protein coupled receptor 40 (GPR40) and p38 MAPK. GPR40/p38 MAPK activation by RGZ was shown in human endothelial cells to markedly augment downstream RGZ/PPARγ genomic signaling (J Biol Chem 2015). This direct connection between GPR40 signaling and PPARγ transcriptional activation argues that PPARγ ligand effects on human endothelium might be best understood as a cognate two-receptor system, integrated by p38 MAPK, PGC1α, and EP300. The MR-independent anti-inflammatory effects of spironolactone have been a source of speculation for decades, but the mechanism has been elusive. Spironolactone, but not eplerenone was found to suppress both NF-κB and AP-1 inflammatory signaling independent of MR. Spironolactone-induced proteasome-degradation of XPB was identified as the mechanism of this potent anti-inflammatory effect (manuscript in preparation 2015). Pulmonary arterial hypertension pathogenesis and therapeutic targets: Heterozygous germline mutations in bone morphogenetic protein type II receptor (BMPR2) are the most common genetic cause of PAH accounting for 70% of familial cases and 10-40% of sporadic idiopathic PAH. However, BMPR2 expression is markedly reduced in end-stage PAH, even in patients not harboring mutations. Loss of ligand-dependent, canonical BMPR2/Smad signaling appears to be important in PAH pathogenesis. However, some mutations in patients with PAH are limited to the cytoplasmic tail of BMPR2, leaving Smad signaling intact. The BMPR2 tail regulates stress kinase pathways and interacts with scaffolding proteins and cytoskeletal components. Importantly, ligand-independent, non-canonical signaling and functional abnormalities are universal across mutant BMPR2 genotypes, but less well understood. Genome-wide gene expression differences in the peripheral blood mononuclear cells (PBMCs) of patients with PAH compared to healthy gender, age and ethnicity matched volunteers categorically identified alterations in inflammation, cell adhesion, cell motility, the cytoskeleton and apoptosis (American Thoracic Society abstract 2011). Specific genes and canonical pathways overlapped with several previously proposed mechanisms of PAH such as Ras, RhoA, integrin, focal adhesion kinase-1 (FAK), and p21-activated kinase (PAK). Circulating endothelial cells (CECs) have been proposed as a biomarker of vascular injury. CECs were identified by flow cytometry and their endothelial phenotype was validated using ultramicro analytical immunochemistry (Thrombosis and Haemostasis 2014). Mutations in the BMPR2 gene leading to loss-of-function are the most common cause of heritable PAH. BMPR2 knockdown (KD) in human pulmonary artery endothelial cells (PAECs) activated the Ras/Raf/ERK signaling pathway leading to proliferation, invasiveness and cytoskeletal abnormalities. Ras/Raf/ERK signaling, an important oncogenic pathway, may contribute to pathologic vascular remodeling in PAH. Therapeutics targeting non-canonical Raf/ERK signaling may be useful in preventing or reversing vascular remodeling in patients with PAH (J Am College Cardiol abstract 2014; manuscript submitted 2015). Two clinical protocols on PAH are now opened for enrollment and provide a source of clinical specimens for ongoing laboratory studies: 1) A Pilot Study of the Effect of Spironolactone Therapy on Exercise Capacity and Endothelial Dysfunction in Pulmonary Arterial Hypertension. This is a randomized, double blinded, placebo-controlled study in patients with PAH of early treatment with spironolactone (Trials 2013). 2) A Natural History Study of Novel Biomarkers in Pulmonary Arterial Hypertension. This study investigates the ability of circulating markers of vascular inflammation as well as high-resolution cardiac magnetic resonance imaging (MRI) to accurately stage severity of disease and/or predict clinically relevant outcomes.

Agency
National Institute of Health (NIH)
Institute
Clinical Center (CLC)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIACL000188-18
Application #
9154033
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
18
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Clinical Center
Department
Type
DUNS #
City
State
Country
Zip Code
Elinoff, Jason M; Chen, Li-Yuan; Dougherty, Edward J et al. (2018) Spironolactone-induced degradation of the TFIIH core complex XPB subunit suppresses NF-?B and AP-1 signalling. Cardiovasc Res 114:65-76
Yang, Zhi-Hong; Gordon, Scott M; Sviridov, Denis et al. (2017) Dietary supplementation with long-chain monounsaturated fatty acid isomers decreases atherosclerosis and alters lipoprotein proteomes in LDLr(-/-) mice. Atherosclerosis 262:31-38
Dougherty, Edward J; Elinoff, Jason M; Ferreyra, Gabriela A et al. (2016) Mineralocorticoid Receptor (MR) trans-Activation of Inflammatory AP-1 Signaling: DEPENDENCE ON DNA SEQUENCE, MR CONFORMATION, AND AP-1 FAMILY MEMBER EXPRESSION. J Biol Chem 291:23628-23644
Wang, Shuibang; Dougherty, Edward J; Danner, Robert L (2016) PPAR? signaling and emerging opportunities for improved therapeutics. Pharmacol Res 111:76-85
Awad, Keytam S; Elinoff, Jason M; Wang, Shuibang et al. (2016) Raf/ERK drives the proliferative and invasive phenotype of BMPR2-silenced pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 310:L187-201
Wang, Shuibang; Awad, Keytam S; Elinoff, Jason M et al. (2015) G Protein-coupled Receptor 40 (GPR40) and Peroxisome Proliferator-activated Receptor ? (PPAR?): AN INTEGRATED TWO-RECEPTOR SIGNALING PATHWAY. J Biol Chem 290:19544-57
Tropea, Margaret M; Harper, Bonnie J A; Graninger, Grace M et al. (2014) Isolation of a circulating CD45-, CD34dim cell population and validation of their endothelial phenotype. Thromb Haemost 112:770-80
Ferreyra, Gabriela A; Elinoff, Jason M; Demirkale, Cumhur Y et al. (2014) Late multiple organ surge in interferon-regulated target genes characterizes staphylococcal enterotoxin B lethality. PLoS One 9:e88756
Elinoff, Jason M; Rame, J Eduardo; Forfia, Paul R et al. (2013) A pilot study of the effect of spironolactone therapy on exercise capacity and endothelial dysfunction in pulmonary arterial hypertension: study protocol for a randomized controlled trial. Trials 14:91
Hicks, Caitlin W; Sweeney, Daniel A; Danner, Robert L et al. (2012) Beneficial effects of stress-dose corticosteroid therapy in canines depend on the severity of staphylococcal pneumonia. Intensive Care Med 38:2063-71

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