Vascular Dysfunction and Inflammation

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Nitric oxide (NO): NO upregulates 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 (FASEB J 2007) protecting the endothelium. Unlike NO, CO blocked NF-kB signaling, broadly suppressing inflammation (PLoS One 2009). Nuclear receptors (NRs): G-protein coupled receptor 40 (GPR40)/p38 MAPK/PGC1a/EP300 activation by rosiglitazone (RGZ) was shown 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). Long-chain monounsaturated fatty acids (LCMUFA; i.e., C20:1 and C22:1) benefits were associated with PPAR activation, via GPR40 activation (Atherosclerosis 2017). 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. 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). CFH infusions resulted in pulmonary hypertension, cardiogenic shock, and multiorgan failure, likely through NO scavenging. During sepsis, CFH infusions worsened oxygen exchange and lung injury, presumably by supplying iron that promoted bacterial growth. CFH elevation in septic shock adversely impacts sepsis outcomes through more than one mechanism that could be therapeutically targeted (Am J Physiol Heart Circ Physiol 2021). 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). 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 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 PBMC expression profiling in PAH patients 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. CAV1 loss and STAT1 activation was also seen in the pulmonary arterioles of patients with idiopathic PAH. Blocking JAK/STAT or AKT rescued aspects of CAV1 loss, only AKT inhibitors suppressed activation of both signaling pathways. 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). MR antagonist treatment in the SuHx rat model of PAH preserved cardiac index and increased left ventricular (LV) end-diastolic volume index. EPL treatment blunted the induction of MR target and inflammatory response genes in the RV (Am J Physiol Lung Cell Mol Physiol 2022). In our CAV1 loss model of PAH, NOS3 co-silencing not only blocked STAT1 and NOS3 phosphorylation, but also diminished ROS generation. Small molecule inhibitors of sAC and PKA blocked NOS3 and STAT1 phosphorylation suggesting a possible role for this pathway in CAV1 loss associated abnormalities (ATS abstract 2022). Neither pseudotyped nor live SARS-CoV-2 virus appear to readily infect or replicate in human pulmonary ECs in vitro, with the exception of the D614G variant. Low expression of ACE2 and TMPRSS2 in PAECs may explain their diminished susceptibility to SARS-CoV-2 (ATS abstract 2022). LOF mutations in COUPTF2 (NR2F2) have been associated with 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 (Am J Physiol Lung Cell Mol Physiol 2023). 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 submission pending 2023). Marked resistance to apoptosis has been a consistent feature of our EC models of PAH. Using the BMPR2 LOF model as a prototype, apoptosis resistance was linked to DLL4/NOTCH1 signaling loss with PI3K/AKT activation, and JNK suppression (Aspen Lung Conference 2019). Importantly, DLL4 loss was seen across several of our models of PAH and validated in lung tissue from iPAH patients. Blocking PI3K/AKT or PPARgamma overexpression restored apoptosis sensitivity in three model systems, BMPR2, CAV1 and PHD2 (ATS abstract 2023). Interactions among BMPR2, DLL4/NOTCH1/PPAR, and PI3K/AKT may lead to targeted approaches for treating vascular remodeling in PAH (MS pending submission 09/2023). Vasohibin-1 (VASH1) loss with increased alpha-tubulin tyrosination was implicated in BMPR2 loss-associated cytoskeletal abnormalities and endothelial dysfunction (MS in preparation 2024). 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). COVID-19 has acute and chronic manifestations. 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 coronavirus mouse model. Preliminary results have demonstrated that SARS-CoV-2 viral proteins are readily taken up by human endothelium and trigger a senescent cellular phenotype (NIH Research Festival 2023; Fellows Award for Research Excellence (FARE) 2024). Endothelial senescence in this model system is reversable using a drug undergoing phase III testing in acute COVID-19. Associate Investigator on a 2023 Bench to Bedside application: Effect of dietary fish oil enriched in very-long-chain polyunsaturated fatty acids (VLCPUFA) on cardiometabolic risk factors and visual function. Principle Investigator on a 2023 Bench to Bedside application: Endothelial senescence in acute and late covid-19 vasculopathy.