Vascular Dysfunction and Inflammation

Clinical Center

Investigators

Linked publications, trials & patents

Abstract

Nitric oxide (NO): NO up-regulates TNFa production (J Immunol 1994; Blood 1997) through a cGMP-independent pathway (J Biol Chem 1997) utilizing NO-responsive Sp1 promoter binding sites (J Biol Chem 1999; J Biol Chem 2003). Dysfunctional eNOS upregulates TNFa (J Biol Chem 2000) through ROS and ERK1/2 (Am J Physiol 2001). NO activation of p38 MAPK stabilizes IL-8 mRNA (J Infect Dis 1998; J Leuk Biol 2004). NO has diverse effects on transcript stability and translation (Nucleic Acids Research 2006; J Leuk Biol 2008). Sickle cell disease causes oxidant and inflammatory stress in the vasculature (Blood, 2004). This circulatory stress alters gene expression and arginine metabolism (Circulation, 2007). NO anti-proliferative effects linked to p38 MAPK activation and p21 mRNA stabilization (BMC Genomics 2005; J Biol Chem 2006). NO and peroxisome proliferator-activated receptors (PPARs) protect endothelium and regulate its function. PPARg is activated by NO through p38 MAPK signaling (FASEB J 2007). In contrast to pro-inflammatory effects of high output NO, CO blocks proximal events in NF-kB signaling, broadly suppressing inflammation (PLoS One 2009). Nuclear receptors (NRs): GR suppresses inflammation by tethering to DNA-bound NF-kB and AP-1 complexes that broadly control the expression of cytokines, chemokines and adhesion molecules. Other NRs including PPARg, MR, AR, and COUP-TF also regulate inflammation in human endothelial cells (ECs). 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 ECs to augment RGZ/PPARg genomic signaling (J Biol Chem 2015). Cognate GPR and nuclear receptor signaling networks may explain differences in the safety and efficacy of NR targeted drugs (Pharm Research 2016). 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 PPAR activation, possibly via the activation of GPR40, and favorable alterations in lipoproteins (Atheroscelerosis 2017). 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, a core subunit of the eukaryotic basal transcription TFIIH complex (Cardiovasc Res 2018). Pulmonary arterial hypertension (PAH): Two 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). ECs with heterogeneous PAH-associated molecular defects including BMPR2, CAV1 and SMAD9, PHD2 (prolyl hydroxylase domain protein 2; EGLN1), COUPTF2 (NR2F2), and G6PC3 (glucose-6- phosphatase catalytic subunit 3) are being studied in vitro to create a comprehensive picture of pathogenic mechanisms and therapeutic targets. 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 oncogenic pathway, leading to proliferation, invasiveness and cytoskeletal abnormalities (Am J Physiol Lung Cell Mol Physiol 2016). A meta-analysis of peripheral blood mononuclear cell (PBMC) expression profiling in PAH patients from multiple centers and across various expression profiling platforms identified an interferon-driven systemic immunologic response as a fundamental component of PAH pathobiology that was previously unrecognized in the individual blood expression profiling studies (Am J Physiol Lung Cell Mol Physiol 2020). Caveolin-1 (CAV1) loss-of-function (LOF), similar to BMPR2, produced a proliferative, hyper- migratory and inflammatory PAEC phenotype (Grover Conference 2015; ATS 2017) with activation of JAK/STAT/interferon signaling and AKT. This inflammatory signature was also found in fibroblasts from PAH patients with CAV1 mutations and in CAV1-/- mice (Aspen Lung Conference 2019; ATS 2017). Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 frameshift mutations. While blocking JAK/STAT or AKT rescued aspects of CAV1 loss, only AKT inhibitors suppressed activation of both signaling pathways simultaneously. Silencing endothelial nitric oxide synthase (NOS3) prevented STAT1 and AKT activation induced by CAV1 loss, implicating CAV1/NOS3 uncoupling and NOS3 dysregulation in the inflammatory phenotype associated with CAV1 loss (Proc Natl Acad Sci U S A 2021). A sugen (SU5416) hypoxia rat model of pulmonary arterial hypertension has been established and an initial study of spironolactone and eplerenone compared to placebo has been completed (AHA Meeting 2019; MS submitted 2021). Loss-of-function mutations in COUPTF2 (NR2F2) have been associated with congenital heart disease (CHD), which can result in PAH. COUPTF2 silencing in ECs produced an interferon inflammatory response and exhibited a hyper-proliferative, apoptosis-resistant, and invasive phenotype with AKT activation. Dickkopf-1 (DKK1), an upstream regulator of AKT, was induced by COUPTF2 silencing and DKK1 knockdown abrogated the abnormal signaling associated with COUPTF2 loss (Aspen Lung Conference 2019: MS in preparation). SMAD9 LOF in human PAECs also produced an abnormal cellular phenotype characterized by proliferation, hypermigration, cytoskeletal and mitochondrial alterations and endothelial to mesenchymal transition, as well as non-canonical activation of AKT, ERK and p38 (ATS 2018; MS in preparation). An in vitro pseudohypoxia model of PAH was established by silencing PHD2 (prolyl hydroxylase domain protein 2; EGLN1) in LMVECs. PHD2-silencing stabilized HIF2alpha, decreased ASK- interacting protein 1 (AIP; DAB2IP), and activated AKT and ERK (Aspen Lung Conference 2019; MS in preparation). Marked resistance to apoptosis has been a consistent feature of our endothelial cell models of PAH. Using the BMPR2 loss-of-function model as a prototype, apoptosis resistance was linked to vasohibin 1 (VASH1) and DLL4 loss, PI3K/AKT and ERK activation, and JNK suppression, (Aspen Lung Conference 2019: MS in preparation). Inhibiting PI3K/AKT restored apoptosis sensitivity in the three model systems tested to date, BMPR2, CAV1 and PHD2. More recently, increased alpha-tubulin tyrosination was implicated in BMPR2 loss-associated endothelial dysfunction. Severe cardiovascular complications, major thrombotic events and widespread organ injury from microvascular disease contribute to the morbidity and mortality of COVID-19. As such, understanding the mechanisms by which SARS-CoV-2 causes endothelial dysfunction and injury in myriad vascular beds is necessary to prevent or treat COVID-19 vasculopathy. To address this unmet need, we are investigating the effects of ACE2 and CD147 loss on human pulmonary artery endothelial cell (PAEC) function. Preliminary findings suggest that ACE2 and CD147 loss in severe COVID-19 results in a thrombogenic vasculopathy secondary to an inflammatory and dysfunctional endothelium.