NO production by endothelial nitric oxide synthase (eNOS; NOS-3) is cytoprotective and regulates smooth muscle tone, leukocyte adhesion, and platelet aggregation. In contrast, inducible NOS (iNOS; NOS-2) during sepsis produces large amounts of NO resulting in shock, myocardial depression, tissue injury and apoptosis. Our work has focused on cGMP-independent, non-canonical NO signaling and inflammatory gene regulation. NO up-regulated TNFa 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 upregulated TNFa (J Biol Chem 2000) through ROS and ERK1/2 (Am J Physiol 2001). NO activation of p38 MAPK stabilized IL-8 mRNA (J Infect Dis 1998; J Leuk Biol 2004). Additional mechanisms demonstrated the diversity of NO effects on transcript stability and translation (Nucleic Acids Research 2006; J Leuk Biol 2008). Sickle cell disease caused oxidant and inflammatory stress in the vasculature (Blood, 2004). This circulatory stress 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 (BMC Genomics 2005; J Biol Chem 2006). Both NO and peroxisome proliferator-activated receptors (PPARs) protect the endothelium and regulate its function. PPARg was activated by NO through a p38 MAPK signaling pathway (FASEB J 2007). In contrast to the pro-inflammatory effects of high output NO, CO blocked proximal events in NF-kB signal transduction and broadly suppressed inflammation (PLoS One 2009). Nuclear receptors (NRs): Glucocorticoid (GC) activation of GR is used extensively to treat inflammation. In addition to inducing anti-inflammatory mediators, GR suppresses inflammatory responses by tethering to DNA-bound NF-kB and AP-1 complexes, transcription factor families that broadly control the expression of cytokines, chemokines and adhesion molecules. This trans-repression mechanism has also been described for some of the other 47 human NRs. Regulatory effects of PPARg, MR, AR, and COUP-TF on inflammation are being investigated in human endothelium. Rosiglitazone (RGZ) is a PPARg ligand/agonist used to treat type 2 diabetes. G-protein coupled receptor 40 (GPR40)/p38 MAPK/PGC1a/EP300 activation by RGZ was shown in human endothelial cells (ECs) to augment RGZ/PPARg genomic signaling (J Biol Chem 2015). Thus, RGZ effects are best understood as a cognate two-receptor system, integrated by p38 MAPK, PGC1a, and EP300 (Pharm Research 2016). In human ECs, MR agonists repressed NF-kB mediated gene transcription, but trans-activated inflammatory AP-1 signaling in a DNA sequence, MR conformation, and AP-1 family member dependent fashion (J Biol Chem 2016). Aldosterone/MR activation of AP-1 may contribute to harmful inflammatory effects in CHF and PAH. Long-chain monounsaturated fatty acids (LCMUFA; i.e., C20:1 and C22:1) benefits were associated with the activation of Ppar signaling pathways, possibly via the activation of GPR40, and favorable alterations in the proteome of lipoproteins (Atheroscelerosis 2017). The MR-independent anti-inflammatory effects of spironolactone (SPL) have been poorly understood. SPL, but not eplerenone was found to suppress both NF-kB and AP-1 inflammatory signaling independent of MR through the proteasomal degradation of XPB (Cardiovasc Res 2018). COUPTFII (gene symbol: NR2F2) loss-of-function mutations cause congenital heart defects and PAH, presumably from increased pulmonary vascular shear stress. Loss of COUPTF2 was also found to increase interferon signaling, AKT, ERK, and mTOR phosphorylation and endothelial cell proliferation suggesting a predisposition PAH independent of structural cardiac abnormalities (in preparation). Selective AR modulators (SARMs) supplied by GSK were investigated and compared to dihydrotestosterone (DHT) to identify AR ligands with less pro-inflammatory potential and possibly net anti-inflammatory effects in the human vasculature. (in preparation). Pulmonary arterial hypertension pathogenesis and therapeutic targets: Two PAH clinical protocols, including a pilot study of spironolactone therapy (Trials 2013) and a natural history study investigating circulating markers of vascular inflammation and high-resolution cardiac magnetic resonance imaging (MRI), provide a source of patient specimens to support ongoing laboratory studies. Circulating ECs were identified by flow cytometry and their endothelial phenotype was validated using ultramicro analytical immunochemistry (Thrombosis and Haemostasis 2014). Gene expression differences in the peripheral blood mononuclear cells (PBMCs) of patients with PAH compared to healthy gender, age and ethnicity matched volunteers identified alterations in inflammation, cell adhesion, cell motility, the cytoskeleton and apoptosis (American Thoracic Society abstract 2011). A meta-analysis was performed of PAH/PBMC expression profiling studies from multiple centers and across various expression profiling platforms (ATS abstract 2016; manuscript in preparation). The in vitro profiling of ECs with heterogeneous PAH-associated molecular defects, such as those involving BMPR2, CAV1 and SMAD9 are being studied to create a comprehensive picture of pathogenic mechanisms and therapeutic targets. Heterozygous loss-of-function mutations in bone morphogenetic protein type II receptor (BMPR2) are the most common genetic cause of PAH. BMPR2 knockdown (KD) in human pulmonary artery ECs (PAECs) activated Ras/Raf/ERK signaling, an important oncogenic pathway, leading to proliferation, invasiveness and cytoskeletal abnormalities (Am J Physiol Lung Cell Mol Physiol 2016). Caveolin-1 (CAV1) loss-of-function (LOF) was found, similar to BMPR2, to produce a proliferative, hyper-migratory and inflammatory PAEC phenotype (Grover Conference 2015) with activation of JAK/STAT/interferon signaling (in preparation). Dermal fibroblasts from PAH patients with CAV1 mutations displayed this same abnormal cellular phenotype (ATS abstract 2017). SMAD9 LOF in human PAECs produces an abnormal cellular phenotype similar to BMPR2 silencing. Notably, all three in vitro models, BMPR2, CAV1 and SMAD9, show universal AKT activation, which is being explored as a possible therapeutic target to block pathologic vascular remodeling across heterogeneous PAH-associated genotypes. Serial cardiac MRI in the Su-5416/hypoxia/normoxia rat model of PAH demonstrated significant right ventricular dysfunction at week 5, providing a useful measure to follow disease progression and therapeutic responses (ATS abstract 2017). EC apoptosis resistance was investigated and several therapeutic targets were identified for reversing pathologic vascular remodeling in PAH (in preparation). An in vitro pseudohypoxia model of PAH was established by silencing PHD2 (prolyl hydroxylase domain protein 2) in human lung microvascular endothelial cells. PHD2-silencing activated AKT and ERK, but inactivated JNK driving a proliferative and apoptosis resistant cellular phenotype.
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