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; J Biol Chem 1999; J Biol Chem 2003), while ROS from eNOS uncoupling upregulates TNFa (J Biol Chem 2000) through 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), altering gene expression and arginine metabolism (Circulation, 2007). NO activation of p38 MAPK stabilized p21 mRNA and was antiproliferative (BMC Genomics 2005; J Biol Chem 2006). NO activated PPARg through p38 MAPK signaling (FASEB J 2007) protecting the endothelium and regulating its function. Unlike the pro-inflammatory effects of 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 may also regulate inflammation in human endothelial cells (ECs). G-protein coupled receptor 40 (GPR40)/p38 MAPK/PGC1a/EP300 activation by Rosiglitazone (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 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 contribute to harm in CHF and PAH. Long-chain monounsaturated fatty acids (LCMUFA; i.e., C20:1 and C22:1) benefits were associated with PPAR activation, via GPR40 activation (Atheroscelerosis 2017). Spironolactone (SPL) suppresses both NF-kB and AP-1 inflammatory signaling independent of MR through proteasomal degradation of XPB, a subunit of the TFIIH transcription complex (Cardiovasc Res 2018). Pulmonary arterial hypertension (PAH): A pilot study of SPL therapy (Trials 2013) and a natural history study investigating vascular inflammation support ongoing laboratory studies. Circulating ECs were identified and validated using flow cytometry and ultramicro analytical immunochemistry (Thromb Haemostasis 2014). ECs with 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 to uncover 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 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 platforms identified IFN-driven inflammation as a fundamental component of PAH pathobiology that was unrecognized in individual blood 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 associated with JAK/STAT/interferon and AKT activation. This inflammatory signature was also found in fibroblasts from PAH patients with CAV1 mutations and in CAV1-/- mice. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH. 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 in the inflammatory phenotype associated with CAV1 loss (Proc Natl Acad Sci USA 2021). Utilizing serial, high-resolution cardiac cine magnetic resonance imaging (MRI), MR antagonist treatment beginning after week 5 in the SuHx rat model of PAH preserved cardiac index and increased left ventricular (LV) end-diastolic volume index. Consistent with these beneficial effects on myocardial function, EPL treatment blunted the induction of MR target and inflammatory response genes in the RV (Am J Physiol Lung Cell Mol Physiol 2022). 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 IFN inflammatory response and a hyper-proliferative, apoptosis-resistant, and invasive phenotype. Dickkopf-1 (DKK1) was induced by COUPTF2 loss and DKK1 knockdown abrogated signaling and phenotypic abnormalities (MS submitted 2022). 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 DLL4 loss with dysregulated NOTCH1 signaling resulting in PI3K/AKT and ERK activation, and JNK suppression (Aspen Lung Conference 2019). Importantly, DLL4 loss was seen across several of our in vitro models of PAH and validated in lung tissue from patients with iPAH (MS in preparation 2022). Blocking PI3K/AKT with a small molecule inhibitor or PPARgamma overexpression restored apoptosis sensitivity in three model systems, BMPR2, CAV1 and PHD2. More recently, vasohibin-1 (VASH1) loss with increased alpha-tubulin tyrosination was implicated in BMPR2 loss-associated cytoskeletal abnormalities and endothelial dysfunction (MS in preparation 2023). COVID-19 has both acute and chronic manifestations. Acute severe disease is associated with multiorgan failure and small vessel vasculopathy characterized by widespread microthrombi. Long-term survivors have an increased risk for cardiovascular complications including stroke and myocardial infarction (MI). Endothelial senescence may underlie the thrombotic microvasculopathy associated with severe COVID-19 as well as the increased risk of cardiovascular events in patients who have otherwise recovered. We have launched a multi-institute project to investigate this hypothesis using deeply phenotyped patient cohorts, a bioengineered three-dimensional disease-on-chip in vitro system and a beta-coronavirus mouse model that closely recapitulates key aspects of human disease. Preliminary results have demonstrated that SARS-CoV-2 viral proteins, at concentrations found during severe COVID-19, are readily taken up by human endothelium and trigger a senescent cellular phenotype.