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 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 was found to upregulate TNFa (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. PPARg 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-kB 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 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 now been described for a growing number of other NRs. PPARg, MR, AR, and COUP-TF are prominently expressed in human endothelium. In human endothelial cells, MR agonists were found to repress NF-kB 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 (submitted 2016). Rosiglitazone (RGZ), a PPARg ligand/agonist, also activates G-protein coupled receptor 40 (GPR40) and p38 MAPK. GPR40/p38 MAPK/ PGC1a/EP300 activation by RGZ was shown in human endothelial cells to markedly augment downstream RGZ/PPARg genomic signaling (J Biol Chem 2015). Therefore, PPARg ligand effects on human endothelium might be best understood as a cognate two-receptor system, integrated by p38 MAPK, PGC1a, 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-kB and AP-1 inflammatory signaling independent of MR. Spironolactone-induced proteasomal degradation of XPB was identified as the mechanism of this potent anti-inflammatory effect (in preparation 2016). Pulmonary arterial hypertension pathogenesis and therapeutic targets: Heterozygous loss-of-function 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. Furthermore, BMPR2 expression is markedly reduced in end-stage PAH, even in patients not harboring these mutations. While some BMPR2 mutations associated with PAH do not disrupt canonical BMPR2/Smad signaling, 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 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). BMPR2 knockdown (KD) in human pulmonary artery endothelial cells (PAECs) activated the Ras/Raf/ERK signaling, an important oncogenic pathway, leading to proliferation, invasiveness and cytoskeletal abnormalities. Therapeutics targeting this non-canonical may be useful in preventing or reversing vascular remodeling in patients with PAH (Am J Physiol Lung Cell Mol Physiol 2016). Two clinical protocols on PAH continue to enroll patients with PAH 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. Another genetic defect associated with PAH, caveolin-1 (CAV1) loss-of-function was found, similar to BMPR2, to produce a proliferative, hyper-migratory and inflammatory cellular phenotype, but in this case with abnormalities in interferon signaling (in preparation 2016). Notably, interferon administration and CANDLE syndrome, an interferon-driven auto-inflammatory disease, have both been associated with the development of PAH. The in vitro profiling of endothelial cells with heterogeneous PAH-associated molecular defects, such as those involving BMPR2 and CAV1 can help create a more comprehensive picture of pathogenic mechanisms and therapeutic targets. The comparative biology of seemingly unrelated molecular defects may lead to both individualized and possibly universal approaches for arresting or even reversing pathologic vascular remodeling.
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