Targeting MicroRNA-192-5p, a Downstream Effector of NOXs (NADPH Oxidases), Reverses Endothelial DHFR (Dihydrofolate Reductase) Deficiency to Attenuate Abdominal Aortic Aneurysm Formation

Abstract

We have shown that endothelial-specific DHFR (dihydrofolate reductase) deficiency underlies eNOS (endothelial NO synthase) uncoupling and formation of abdominal aortic aneurysm (AAA). Here, we examined a novel role of microRNA-192-5p in mediating NOX (NADPH oxidase)-dependent DHFR deficiency and AAA formation. microRNA-192-5p is predicted to target DHFR. Intriguingly, homo sapiens–microRNA-192-5p expression was substantially upregulated in human patients with AAA. In human aortic endothelial cells exposed to hydrogen peroxide (H 2 O 2 ), homo sapiens–microRNA-192-5p expression was significantly upregulated. This was accompanied by a marked downregulation in DHFR mRNA and protein expression, which was restored by homo sapiens–microRNA-192-5p–specific inhibitor. Of note, microRNA-192-5p expression was markedly upregulated in Ang II (angiotensin II)–infused hph-1 (hyperphenylalaninemia 1) mice, which was attenuated in hph-1–NOX1, hph-1–NOX2, hph-1–neutrophil cytosol factor 1, and hph-1–NOX4 double mutant mice where AAA incidence was also abrogated, indicating a downstream effector role of microRNA-192-5p following NOX activation. In vivo treatment with mus musculus–microRNA-192-5p inhibitor attenuated expansion of abdominal aortas in Ang II–infused hph-1 mice as defined by ultrasound and postmortem inspection. It also reversed features of vascular remodeling including matrix degradation, adventitial hypertrophy, and formation of intraluminal thrombi. These animals had restored DHFR mRNA and protein expression, attenuated superoxide production, recoupled eNOS, and preserved NO bioavailability. In conclusion, our data for the first time demonstrate a critical role of microRNA-192-5p in mediating NOX-dependent DHFR deficiency and AAA formation, inhibition of which is robustly effective in attenuating development of AAA. Since the mouse and human microRNA-192-5p sequences are identical, the microRNA-192-5p inhibitors may be readily translatable into novel therapeutics for the treatment of AAA.

Therapeutic application of estrogen for COVID-19: Attenuation of SARS-CoV-2 spike protein and IL-6 stimulated, ACE2-dependent NOX2 activation, ROS production and MCP-1 upregulation in endothelial cells

“…Primary bovine aortic endothelial cells (BAECs, Genlantis, passage 4 to 6) were cultured in M199 supplemented with 10% fetal bovine serum (FBS), 1% vitamin, and 1% l -glutamine as previously described [ [36] , [37] , [38] , [39] ]. Primary human aortic endothelial cells (HAECs, Lonza, passage 4 to 6) were cultured in EGM-2 media supplemented with 10% fetal bovine serum and EGM-2 SingleQuots (#CC-4176, Lonza) as previously reported [ 40 ]. Confluent BAECs were starved overnight with 5% FBS in M199 and treated with different concentrations of cigarette smoke extract (CSE, 0, 2.5%, 5%, 10%), recombinant human interleukin-6 (IL-6, 100 ng/mL, #206-IL-010, R&D), or recombinant SARS CoV-2 Spike protein (S protein, 500 ng/mL, #10549-CV-100, R&D) for 24 hrs before harvested for the following experiment.…”

Novel and robust treatment of pulmonary hypertension with netrin-1 and netrin-1-derived small peptides

“…Electron spin resonance (ESR) was used as a gold standard to examine superoxide aninon production and eNOS uncoupling activity as we previously published [19 , 21 , 22 , 24 , 25 , [27] , [28] , [29] , [30] , [31] , [32] , [40] , [41] , [42] , [43] ]. Under normal conditions when eNOS is coupled, the addition of the NOS inhibitor l -NAME will increase the measured superoxide production, as eNOS is producing NO to scavenge superoxide; however, when eNOS is uncoupled and producing superoxide, its inhibition will lead to a decrease in measured superoxide.…”

“…Cryostat transverse cuts (5 μm) of lung sections were freshly prepared at -20°C. Levels of ROS in mouse lungs were determined using dihydroethidium (DHE) fluorescent imaging as we previously published [ 19 , 39 , 40 ]. DHE stock solution was prepared by dissolving DHE (MilliporeSigma, Cat# D7008, St. Louis, MO, USA) in dimethylsulfoxide (DMSO) at a concentration of 2 mM.…”

“…Protein content of the supernatant was determined using a DC TM protein assay kit (Bio-Rad, Cat# 500-0114 for reagent B, Cat# 500-0113 for reagent A, Cat# 400-0115 for reagent S, Hercules, CA, USA). eNOS uncoupling activity was determined from lung homogenates using ESR as we previously published [ 19 , 21 , 22 , 24 , 25 , [27] , [28] , [29] , [30] , [31] , [32] , [40] , [41] , [42] , [43] ]. Total superoxide production was calculated from the SOD-inhibitable fraction.…”

Paeoniflorin Suppresses TBHP-Induced Oxidative Stress and Apoptosis in Human Umbilical Vein Endothelial Cells via the Nrf2/HO-1 Signaling Pathway and Improves Skin Flap Survival

“…Previous studies have reported that the activation of Nrf2/HO-1/HIF-1α axis could upregulate the VEGF expression ( Li et al, 2016 ; Huang et al, 2018 ). Unfortunately, oxidative stress is generated at the injury site under the conditions of oxidative phosphorylation, which is detrimental to endothelial cells ( Huang K. et al, 2021 ). Hence, protecting endothelial cells against oxidative stress is crucial and urgent for the skin flap viability.…”