Endothelial Dysfunction and Vascular Inflammation

Clinical Center

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Abstract

Human monoblastoid U937 cells transfected with inducible nitric oxide synthase (NOS2) produced increased amounts of TNF, demonstrating that NO, in addition to reducing vascular tone, augments the inflammatory response (Blood, 1997). Endothelial NOS (eNOS) was also shown to upregulate TNF (J Biol Chem, 2000). However, eNOS regulated inflammatory responses through both NO (J Immunol, 1994; J Biol Chem, 1997) and reactive oxygen species-based signaling (J Biol Chem, 2000). Superoxide produced by eNOS was shown to upregulate TNF via p42/44 MAPK activation (J Biol Chem, 2001). Sp1 was identified as a bidirectional NO sensor, down regulating eNOS in endothelial cells and upregulating TNF, both via proximal Sp promoter-binding sites (J Biol Chem, 2003). Sickle cell disease (SCD) was characterized by oxidant and inflammatory stress in the vasculature (Blood, 2004). Circulatory stress in SCD was associated with gene expression and arginine metabolism abnormalities in platelets (Circulation, 2007). The anti-proliferative effects of NO were linked to p38 MAPK activation, p21 mRNA stabilization and subsequent down regulation of polo-like kinase 1 through a CDE/CHR promoter site (BMC Genomics, 2005; J Biol Chem, 2006). Both NO and peroxisome proliferator-activated receptors (PPARs) protect the endothelium and regulate its function. We found that PPARgamma was activated by NO in human endothelial cells through a p38 MAPK dependent signaling pathway (FASEB J, 2007). This mechanism may contribute to the anti-inflammatory and cytoprotective effects of NO in the vasculature. The optimal activation of PPARgamma by thiazolidinediones (TZDs) was found to similarly require p38 MAPK activation. TZDs, like homeostatic NO, reduce markers of cardiovascular inflammation. Rosiglitazone (RGZ) activation of p38 MAPK with downstream enhancement of PPARgamma signaling was linked to G-protein coupled receptor 40 (submitted 2014).. Cognate GPR and nuclear receptor signaling networks may explain differences in safety and efficacy among nuclear receptor ligands. Interrogation of our microarray database indicated that mineralocorticoid receptor (MR), androgen receptor (AR), COUP-TFI and COUP-TFII were highly expressed in human endothelial cells compared to leukocytes. Therefore, these nuclear receptors are being studied as potential targets for modulating endothelium inflammation. Using genome-wide microarrays, COUP-TFII knockdown in a human endothelial cell line was shown to modulate 36% of all TNF-responsive genes (American Thoracic Society, abstract 2011). COUP-TFII suppression of TNFlapha-induced genes was associated with interferon signaling pathways, while COUP-TFII amplification of TNFalpha responses was linked to NF-kappaB (manuscript in preparation). In human endothelial cells both MR agonists and antagonists were found to repress NF-kappaB mediated gene transcription (Keystone Symposia on Nuclear Receptors, abstract, 2010; American Thoracic Society, abstract 2010). In contrast, MR appears to either weakly repress or further activate AP1 signaling depending on the gene target. Unexpectedly, spironolactone suppressed both NF-κB and AP-1 inflammatory signaling independent of MR. These findings have implications for the short-term use of mineralocorticoids in septic shock and long-term, early use of spironolactone in PAH (manuscript in preparation). Gene expression differences in the peripheral blood mononuclear cells (PBMCs) of patients with PAH compared to gender and age matched volunteers demonstrate alterations in inflammation, cell adhesion, cell motility, the cytoskeleton and apoptosis (American Thoracic Society, abstract 2011). Upstream regulator and promoter level analyses associated AP1 signaling pathways with this PBMC/PAH signature. Spironolactone, an MR and AR antagonist undergoing clinical evaluation in PAH (see below) was found in vitro to suppress the expression of selected transcripts associated with PAH (manuscript in preparation). PAH plasma-treated human pulmonary artery endothelial cells (PAECs) differentially expressed over 200 uniquely annotated transcripts using an FDR cutoff of 10%. Unexpectedly, 20% of this signature overlapped with BMPR2 silencing in PAECs, an in vitro model of hereditary PAH (see below). More than 90% of this overlap was directionally discordant, suggesting that circulating factors may work to counter genotypic and phenotypic abnormalities that drive PAH. 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) activates the Ras/ Raf/ERK/AP1 signaling pathway leading to cytoskeletal abnormalities, invasiveness, proliferation and inflammation. These phenotypic abnormalities as well as ERK activation and AP1 mediated gene expression were prevented in vitro with Raf inhibitors and the multikinase inhibitor nintedanib (manuscript in preparation). Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. A CEC population in healthy adult human subjects was identified by flow cytometry and confirmed to possess an endothelial phenotype by ultramicro analytical immunochemistry (Thrombosis and Haemostasis 2014). The majority of CECs in healthy individuals have shed their CD146 surface marker and are senescent without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification. Two clinical protocols on PAH are now opened for enrollment, providing a source of clinical specimens for our 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. 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.