Abstract 15092: The Yin-yang of Bmpr2 and Ces1 in the Pulmonary Endothelium Aad Its Role in Pulmonary Arterial Hypertension

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Stuti Agarwal ◽  
Ananya Chakraborty ◽  
David Condon ◽  
Salvador Tello ◽  
Karthik Suresh ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Redox Balance ◽  
Tube Formation ◽  
Vascular Lesions ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Pulmonary Endothelium ◽  
Pulmonary Arterial

Background/Hypothesis: Pulmonary Arterial Hypertension (PAH) is a life-threatening disease characterized by loss of pulmonary microvessels and vascular remodeling. Loss of function BMPR2 mutations contribute to pulmonary endothelial cell (EC) apoptosis and oxidative stress, but their reduced penetrance suggests need for additional modifiers. Carboxylesterase1 (CES1) is endoplasmic reticulum (ER) enzyme responsible for detoxification, proteostasis, and redox balance. Similar to BMPR2 mutations, we found that loss of CES1 is associated with oxidative stress and apoptosis. In this study, we explore a plausible link between BMPR2 pathway and CES1 regulation in promoting EC survival and angiogenesis. Methods: PECs & lung tissue from healthy donors and PAH patients were obtained from PHBI. To induce oxidative stress, we used H 2 O 2 (100 μM) & methamphetamine HCl (METH, 0.5-2mM). Both siRNA and pharmacological approaches were used to inhibit BMPR2, Nrf2, and CES1 expression. Caspase and Matrigel assays were used to assess PEC survival and tube formation, respectively. Results: RNAseq of BMPR2-mutant PECs showed significantly less CES1 expression, which correlated with reduced protein expression in PEC lysates and within lung vascular lesions. In healthy PECs, BMP9 stimulation led to increase in CES1 expression that was absent post BMPR2 knockdown. CES1 gene transcription was by BMPR2-dependent activation of Nrf2, a transcription factor responsible for antioxidant gene expression and mitochondrial biogenesis. Inhibition of Nrf2 activation by ML385 (5μM) abrogates BMP9 induced CES1 mRNA levels similar to BMPR2 knockdown. The connection between BMPR2 and CES1 was further strengthened by CES1 knockdown studies in PECs that demonstrated reduction in BMPR2 protein synthesis associated with ER stress and reduced autophagy. Finally, lung examination in CRISPR generated CES1 +/- mice demonstrated increased microvascular muscularization at normoxia compared to wild type mice. Conclusion: BMPR2 and CES1 are part of common signaling pathway that protects PECs against oxidative stress and mitochondrial damage through a positive feedback loop. Interventions that restore CES1 activity could rescue BMPR2 signaling and serve as novel PAH therapeutics.

Abstract 14631: Pulmonary Artery Calcification: A New Threat in Pulmonary Arterial Hypertension

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Sophie Chabot ◽  
Grégoire Ruffenach ◽  
Virginie F Tanguay ◽  
François Potus ◽  
Steeve Provencher ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Pulmonary Artery ◽  
Arterial Hypertension ◽  
Annexin V ◽  
Vascular Lesions ◽  
Loss Of Function ◽  
Alizarin Red ◽  
Pulmonary Hemodynamic ◽  
Related Transcription Factor ◽  
Pulmonary Arterial

Background: Pulmonary arterial hypertension (PAH) is associated with imbalance between proliferation and apoptosis of pulmonary artery (PA) smooth muscle cells (PASMCs). Recently, we uncovered a role for microRNA-204 (miR-204), whose downregulation sustains the phenotype in time. In systemic vascular disease, miR-204 downregulation increases the Runt-related transcription factor 2 (RUNX2), a key osteogenic factor inducing calcification. The fact that PAH patients live longer increases the odds of developing PA calcification lesions. Interestingly, the presence and development of calcified lesions in PAH have not been explored. We thus hypothesized that miR-204-dependent upregulation of RUNX2 triggers the development of proliferative and calcified vascular lesions within the lungs, thus worsening PAH. Methods/Results: Using CT-scan, we showed that PAH patients (n=50; p<0.05) had significant increase in distal PA calcification compared to control patients matched for age, sex and comorbidities known to promote vascular calcification. This non-invasive finding was histologically confirmed in the same patients post lung transplantation using Von Kossa staining (n=10; p<0.05). Finally, increase in calcification contributes to worsening of PA compliance and is associated with poorer PAH patients’ survival (n=50; p<0.05). At the molecular level, enhanced calcification in PAH patients correlates (n=43; p<0.05) with a miR-204-dependent up regulation of RUNX2 in lungs, distal PA and primary cultured PASMCs (n=12-40, fold change ≈ 2.5). Using gain and loss of function approaches, we demonstrated in PASMCs from PAH and healthy donors (using siRNA and adenoviruses respectively) that RUNX2, promotes PAH-PASMC proliferation (Ki67), resistance to apoptosis (Annexin-V) and calcification (Alizarin red) (p<0.05). Finally, in Sugen/hypoxia rat model of PAH, we demonstrated that RUNX2 inhibition improves pulmonary hemodynamic and reduces PA remodeling and calcification (n=5-12, p<0.05). Conclusion: We demonstrated for the first time the presence of significant calcification in PAH distal PA contributing to PAH worsening and identified RUNX2 as an attractive new therapeutic strategy to improve PAH. *SC and GR equally contributed.

scholarly journals Effects of ovariectomy on antioxidant defence systems in the right ventricle of female rats with pulmonary arterial hypertension induced by monocrotaline

2018 ◽  
Vol 96 (3) ◽  
pp. 295-303 ◽  
Author(s):  
Rafaela Siqueira ◽  
Rafael Colombo ◽  
Adriana Conzatti ◽  
Alexandre Luz de Castro ◽  
Cristina Campos Carraro ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pulmonary Arterial Hypertension ◽  
Right Ventricle ◽  
Protein Expression ◽  
Arterial Hypertension ◽  
Female Rats ◽  
Antioxidant Defenses ◽  
Thioredoxin 1 ◽  
Pulmonary Arterial ◽  
The Right

The aim of this study was to evaluate the impact of ovariectomy on oxidative stress in the right ventricle (RV) of female rats with pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). Rats were divided into 4 groups (n = 6 per group): sham (S), sham + MCT (SM), ovariectomized (O), and ovariectomized + MCT (OM). MCT (60 mg·kg−1 i.p.) was injected 1 week after ovariectomy or sham surgery. Three weeks later, echocardiographic analysis and RV catheterisation were performed. RV morphometric, biochemical, and protein expression analysis through Western blotting were done. MCT promoted a slight increase in pulmonary artery pressure, without differences between the SM and OM groups, but did not induce RV hypertrophy. RV hydrogen peroxide increased in the MCT groups, but SOD, CAT, and GPx activities were also enhanced. Non-classical antioxidant defenses diminished in ovariectomized groups, probably due to a decrease in the nuclear factor Nrf2. Hemoxygenase-1 and thioredoxin-1 protein expression was increased in the OM group compared with SM, being accompanied by an elevation in the estrogen receptor β (ER-β). Hemoxygenase-1 and thioredoxin-1 may be involved in the modulation of oxidative stress in the OM group, and this could be responsible for attenuation of PAH and RV remodeling.

Abstract 382: Cyclophilin A (CypA) is a Pathogenic Mediator of Pulmonary Arterial Hypertension

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Chao Xue
Keyword(s):  
Oxidative Stress ◽  
Smooth Muscle ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Specific Inhibitor ◽  
Cyclophilin A ◽  
Pulmonary Vasculature ◽  
Tunel Staining ◽  
Mesenchymal Transition ◽  
Pulmonary Arterial

Rationale: Pulmonary arterial hypertension (PAH) is a devastating disease in which oxidative stress has been proposed to mediate pathological changes to the pulmonary vasculature such as endothelial cell (EC) apoptosis, endothelial to mesenchymal transition (EndMT), vascular smooth muscle cell (VSMC) proliferation, and inflammation. Our previous study showed that cyclophilin A (CypA) was secreted from EC and VSMC in response to oxidative stress, and much of the secreted CypA was acetylated (AcK-CypA). Furthermore, CypA was increased in the plasma of patients with PAH. Objective: To evaluate the cell- s pecific role of CypA in PAH and compare the relative effects of AcK-CypA and CypA on EC apoptosis, development of an inflammatory EC phenotype and EndMT. Methods and Results: Transgenic overexpression of CypA in EC, but not SMC, caused a PAH phenotype including increased pulmonary artery pressure, α-smooth muscle actin expression in small arteries, and CD45 positive cells in the lungs. Mechanistic analysis using cultured mouse lung microvascular EC showed that CypA and AcK-CypA increased apoptosis measured by caspase 3 cleavage and TUNEL staining. MM284, a specific inhibitor of extracellular CypA, prevented EC apoptosis. In addition, CypA and AcK-CypA promoted an EC inflammatory phenotype assessed by increased VCAM1 and ICAM1 expression, phosphorylation of p65, and degradation of IkB. Furthermore, CypA and AcK-CypA promoted EndMT assayed by change in cell morphology, increased mesenchymal markers and EndMT related transcription factors. At all concentrations, AcK-CypA stimulated greater increases in apoptosis, inflammation and EndMT than CypA. Conclusions: EC-derived CypA (especially AcK-CypA) causes PAH by a presumptive mechanism involving increased EC apoptosis, inflammation and EndMT. Our results suggest that inhibiting extracellular secreted CypA is a novel therapeutic approach for PAH.

Abstract 263: Differential Effects of 17ß-Estradiol and 16a-Hydroxyestrone in Oxidative Stress and Proliferative Responses in Human Pulmonary Artery Smooth Muscle Cells - Implications in Pulmonary Arterial Hypertension

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Katie Y Hood ◽  
Augusto C Montezano ◽  
Margaret R MacLean ◽  
Rhian M Touyz
Keyword(s):  
Oxidative Stress ◽  
Pulmonary Arterial Hypertension ◽  
Cell Growth ◽  
Arterial Hypertension ◽  
Molecular Mechanisms ◽  
Antioxidant Systems ◽  
Ros Generation ◽  
Ros Production ◽  
Differential Effects ◽  
Pulmonary Arterial

Women develop pulmonary arterial hypertension (PAH) more frequently than men. This may relate, in part, to metabolism of 17β-estradiol (E2), leading to formation of the deleterious metabolite, 16α-hydroxyestrone (16α OHE1), which plays a role in the remodelling of pulmonary arteries. Molecular mechanisms whereby 16αOHE1 influences PASMC remodelling are unclear but ROS may be important, since oxidative stress has been implicated in the pathogenesis of PAH. We hypothesised that E2 and 16αOHE1 leads to Nox-induced ROS production, which promotes PASMC damage. Cultured PASMCs were stimulated with either E2 (1nM) or 16αOHE1 (1nM) in the presence/absence of EHT1864 (100μM, Rac1 inhibitor) or tempol (antioxidant; 10μM). ROS production was assessed by chemiluminescence (O2-) and Amplex Red (H2O2). Antioxidants (thioredoxin, peroxiredoxin 1 and NQ01), regulators of Nrf2 (BACH1, Nrf2) and, marker of cell growth (PCNA) were determined by immunoblotting. E2 increased O2- production at 4h (219 ± 30% vs vehicle; p<0.05), an effect blocked by EHT1864 and tempol. E2 also increased H2O2 generation (152 ± 4%; p<0.05). Thioredoxin, NQ01 and peroxiredoxin1 (71 ± 6%; 78 ± 9%; 69 ± 8%; p<0.05 respectively) levels were decreased by E2 as was PCNA expression (72 ± 2%; p<0.05). 16αOHE1 exhibited a rapid (5 min) and exaggerated increase in ROS production (355 ± 41%; p<0.05), blocked by tempol and EHT1864. This was associated with an increase in Nox4 expression (139 ± 11% vs vehicle, p<0.05). 16αOHE1 increased BACH1, (129 ± 3%; p<0.05), a competitor of Nrf2, which was decreased (92 ± 2%). In contrast, thioredoxin expression was increased by 16aOHE1 (154 ± 22%; p<0.05). PCNA (150 ± 5%) expression was also increased after exposure to 16αOHE1. In conclusion, E2 and 16αOHE1 have differential effects on redox processes associated with PASMC growth. Whereas E2 stimulates ROS production in a slow and sustained manner without effect on cell growth, 16αOHE1 upregulates Nox4 with associated rapid increase in ROS generation and downregulation of antioxidant systems, affecting proliferation. Our findings suggest that E2 -derived metabolites may promote a pro-proliferative PASMC phenotype through Nox4-derived ROS generation. These deleterious effects may impact on vascular remodeling in PAH.

scholarly journals Obesity alters oestrogen metabolism and contributes to pulmonary arterial hypertension

2019 ◽  
Vol 53 (6) ◽  
pp. 1801524 ◽  
Author(s):  
Kirsty M. Mair ◽  
Katie Y. Harvey ◽  
Alasdair D. Henry ◽  
Dianne Z. Hillyard ◽  
Margaret Nilsen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pulmonary Hypertension ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Cytochrome P450 1B1 ◽  
Stress Effects ◽  
Endogenous Oestrogen ◽  
Pulmonary Arterial ◽  
Induced Changes ◽  
Species Production

Obesity is a common comorbidity for pulmonary arterial hypertension (PAH). Additionally, oestrogen and its metabolites are risk factors for the development of PAH. Visceral adipose tissue (VAT) is a major site of oestrogen production; however, the influence of obesity-induced changes in oestrogen synthesis and metabolism on the development of PAH is unclear. To address this we investigated the effects of inhibiting oestrogen synthesis and metabolism on the development of pulmonary hypertension in male and female obese mice.We depleted endogenous oestrogen in leptin-deficient (ob/ob) mice with the oestrogen inhibitor anastrozole (ANA) and determined the effects on the development of pulmonary hypertension, plasma oestradiol and urinary 16α-hydroxyestrone (16αOHE1). Oestrogen metabolism through cytochrome P450 1B1 (CYP1B1) was inhibited with 2,2′,4,6′-tetramethoxystilbene (TMS).ob/ob mice spontaneously develop pulmonary hypertension, pulmonary vascular remodelling and increased reactive oxygen species production in the lung; these effects were attenuated by ANA. Oestradiol levels were decreased in obese male mice; however, VAT CYP1B1 and 16αOHE1 levels were increased. TMS also attenuated pulmonary hypertension in male ob/ob mice. Intra-thoracic fat from ob/ob mice and VAT conditioned media produce 16αOHE1 and can contribute to oxidative stress, effects that are attenuated by both ANA and TMS.Obesity can induce pulmonary hypertension and changes in oestrogen metabolism, resulting in increased production of 16αOHE1 from VAT that contributes to oxidative stress. Oestrogen inhibitors are now in clinical trials for PAH. This study has translational consequences as it suggests that oestrogen inhibitors may be especially beneficial in treating obese individuals with PAH.

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