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. In contrast, inducible NOS (iNOS; NOS-2) during sepsis produces large amounts of NO resulting in shock, myocardial depression, tissue injury and apoptosis. This duality underlies the paradox that both under and over production of NO 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 ROS 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 and broadly suppress inflammation (PLoS One 2009). Nuclear receptors: Nuclear receptors (NRs) regulate cardiovascular homeostasis, metabolism and the immune system. 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. We are specifically investigating the regulatory effects of PPARg, MR, AR, and COUP-TF on inflammation 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 markedly augment downstream RGZ/PPARg genomic signaling (J Biol Chem 2015). Thus, RGZ effects on human endothelium is best understood as a cognate two-receptor system, integrated by p38 MAPK, PGC1a, and EP300 (Pharm Research 2016). In human ECs, MR agonists were found to repress NF-kB mediated gene transcription. In contrast, MR was found to trans-activate 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) have been shown to attenuate atherosclerosis development in mouse models. These 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). MR-independent anti-inflammatory effects of spironolactone have been observed 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 (submitted). 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 (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). 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). Specific genes and pathways overlapped with previously proposed mechanisms of PAH such as Ras, RhoA, integrin, focal adhesion kinase-1 (FAK), and p21-activated kinase (PAK). 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. 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. Heterozygous loss-of-function mutations in bone morphogenetic protein type II receptor (BMPR2) are the most common genetic cause of PAH. Furthermore, BMPR2 expression is markedly reduced in end-stage PAH, even in patients not harboring these mutations. 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. 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). 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). Notably, interferon administration and CANDLE syndrome, an interferon-driven auto-inflammatory disease, have both been associated with the development of PAH. Dermal fibroblasts from PAH patients with CAV1 mutations displayed this same abnormal cellular phenotype (ATS abstract 2017). Preliminary work examining SMAD9 LOF in human PAECs has demonstrated an abnormal cellular phenotype closely related to BMPR2 silencing. Notably, all three in vitro models, BMPR2, CAV1 and SMAD9, all show universal AKT activation. AKT activation is therefore 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).
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