Matrix Stiffening Induces a Pathogenic QKI-miR-7-SRSF1 Signaling Axis in Pulmonary Arterial Endothelial Cells

2021 ◽  
Author(s):  
Chen-Shan Chen Woodcock ◽  
Neha Hafeez ◽  
Adam Handen ◽  
Ying Tang ◽  
Lloyd D Harvey ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Rna Binding ◽  
Vascular Diseases ◽  
Vascular Stiffness ◽  
Artery Stiffness ◽  
Signaling Axis ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells ◽  
Splicing Factor 1

Pulmonary arterial hypertension (PAH) refers to a set of heterogeneous vascular diseases defined by elevation of pulmonary arterial pressure (PAP) and pulmonary vascular resistance (PVR), leading to right ventricular (RV) remodeling and often death. Early increases in pulmonary artery stiffness in PAH drive pathogenic alterations of pulmonary arterial endothelial cells (PAECs), leading to vascular remodeling. Dysregulation of microRNAs can drive PAEC dysfunction. However, the role of vascular stiffness in regulating pathogenic microRNAs in PAH is incompletely understood. Here, we demonstrated that extracellular matrix (ECM) stiffening downregulated miR-7 levels in PAECs. The RNA binding protein Quaking (QKI) has been implicated in the biogenesis of miR-7. Correspondingly, we found that ECM stiffness up-regulated QKI, and QKI knockdown led to increased miR-7. Downstream of the QKI-miR-7 axis, the serine and arginine rich splicing factor 1 (SRSF1) was identified as a direct target of miR-7. Correspondingly, SRSF1 was reciprocally up-regulated in PAECs exposed to stiff ECM and was negatively correlated with miR-7. Decreased miR-7 and increased QKI and SRSF1 were observed in lungs from PAH patients and PAH rats exposed to SU5416/hypoxia. Lastly, miR-7 upregulation inhibited human PAEC migration, while forced SRSF1 expression reversed this phenotype, proving that miR-7 depended upon SRSF1 to control migration. In aggregate, these results define the QKI-miR-7-SRSF1 axis as a mechanosensitive mechanism linking pulmonary arterial vascular stiffness to pathogenic endothelial function. These findings emphasize implications relevant to PAH and suggest the potential benefit of developing therapies that target this miRNA-dependent axis in PAH.

scholarly journals Shear stress stimulates nitric oxide signaling in pulmonary arterial endothelial cells via a reduction in catalase activity: role of protein kinase Cδ

2010 ◽  
Vol 298 (1) ◽  
pp. L105-L116 ◽  
Author(s):  
Sanjiv Kumar ◽  
Neetu Sud ◽  
Fabio V. Fonseca ◽  
Yali Hou ◽  
Stephen M. Black
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Catalase Activity ◽  
Serine Phosphorylation ◽  
Nitric Oxide Signaling ◽  
Enos Uncoupling ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Previous studies have indicated that acute increases in shear stress can stimulate endothelial nitric oxide synthase (eNOS) activity through increased PI3 kinase/Akt signaling and phosphorylation of Ser1177. However, the mechanism by which shear stress activates this pathway has not been adequately resolved nor has the potential role of reactive oxygen species (ROS) been evaluated. Thus, the purpose of this study was to determine if shear-mediated increases in ROS play a role in stimulating Ser1177 phosphorylation and NO signaling in pulmonary arterial endothelial cells (PAEC) exposed to acute increases in shear stress. Our initial studies demonstrated that although shear stress did not increase superoxide levels in PAEC, there was an increase in H2O2 levels. The increases in H2O2 were associated with a decrease in catalase activity but not protein levels. In addition, we found that acute shear stress caused an increase in eNOS phosphorylation at Ser1177 phosphorylation and a decrease in phosphorylation at Thr495. We also found that the overexpression of catalase significantly attenuated the shear-mediated increases in H2O2, phospho-Ser1177 eNOS, and NO generation. Further investigation identified a decrease in PKCδ activity in response to shear stress, and the overexpression of PKCδ attenuated the shear-mediated decrease in Thr495 phosphorylation and the increase in NO generation, and this led to increased eNOS uncoupling. PKCδ overexpression also attenuated Ser1177 phosphorylation through a posttranslational increase in catalase activity, mediated via a serine phosphorylation event, reducing shear-mediated increases in H2O2. Together, our data indicate that shear stress decreases PKCδ activity, altering the phosphorylation pattern catalase, leading to decreased catalase activity and increased H2O2 signaling, and this in turn leads to increases in phosphorylation of eNOS at Ser1177 and NO generation.

scholarly journals Incremental Experience in In Vitro Primary Culture of Human Pulmonary Arterial Endothelial Cells Harvested from Swan-Ganz Pulmonary Arterial Catheters

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3229
Author(s):  
Birger Tielemans ◽  
Leanda Stoian ◽  
Allard Wagenaar ◽  
Mathias Leys ◽  
Catharina Belge ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Diseases ◽  
Right Heart Catheterization ◽  
Heart Catheterization ◽  
Endothelial Phenotype ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells ◽  
Pulmonary Vascular Diseases

Pulmonary arterial hypertension (PAH) is a devastating condition affecting the pulmonary microvascular wall and endothelium, resulting in their partial or total obstruction. Despite a combination of expensive vasodilatory therapies, mortality remains high. Personalized therapeutic approaches, based on access to patient material to unravel patient specificities, could move the field forward. An innovative technique involving harvesting pulmonary arterial endothelial cells (PAECs) at the time of diagnosis was recently described. The aim of the present study was to fine-tune the initial technique and to phenotype the evolution of PAECs in vitro subcultures. PAECs were harvested from Swan-Ganz pulmonary arterial catheters during routine diagnostic or follow up right heart catheterization. Collected PAECs were phenotyped by flow cytometry and immunofluorescence focusing on endothelial-specific markers. We highlight the ability to harvest patients’ PAECs and to maintain them for up to 7–12 subcultures. By tracking the endothelial phenotype, we observed that PAECs could maintain an endothelial phenotype for several weeks in culture. The present study highlights the unique opportunity to obtain homogeneous subcultures of primary PAECs from patients at diagnosis and follow-up. In addition, it opens promising perspectives regarding tailored precision medicine for patients suffering from rare pulmonary vascular diseases.

scholarly journals Role of Carnitine Acetyl Transferase in Regulation of Nitric Oxide Signaling in Pulmonary Arterial Endothelial Cells

2012 ◽  
Vol 14 (1) ◽  
pp. 255-272 ◽  
Author(s):  
Shruti Sharma ◽  
Xutong Sun ◽  
Saurabh Agarwal ◽  
Ruslan Rafikov ◽  
Sridevi Dasarathy ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Signaling ◽  
Carnitine Acetyl Transferase ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Caspase-4/11–Mediated Pulmonary Artery Endothelial Cell Pyroptosis Contributes to Pulmonary Arterial Hypertension

Hypertension ◽  
2022 ◽  
Author(s):  
Yusi Wu ◽  
Bingjie Pan ◽  
Zhen Zhang ◽  
Xiaohui Li ◽  
Yiping Leng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Right Ventricular ◽  
Vascular Remodeling ◽  
Endothelial Cell Function ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Background: Endothelial dysfunction enhances vascular inflammation, which initiates pulmonary arterial hypertension (PAH) pathogenesis, further induces vascular remodeling and right ventricular failure. Activation of inflammatory caspases is an important initial event at the onset of pyroptosis. Studies have shown that caspase-1–mediated pyroptosis has played a crucial role in the pathogenesis of PAH. However, the role of caspase-11, another inflammatory caspase, remains to be elucidated. Therefore, the purpose of this study was to clarify the role of caspase-11 in the development of PAH and its mechanism on endothelial cell function. Methods: The role of caspase-11 in the progression of PAH and vascular remodeling was assessed in vivo. In vitro, the effect of caspase-4 silencing on the human pulmonary arterial endothelial cells pyroptosis was determined. Results: We confirmed that caspase-11 and its human homolog caspase-4 were activated in PAH animal models and TNF (tumor necrosis factor)-α–induced human pulmonary arterial endothelial cells. Caspase-11 −/− relieved right ventricular systolic pressure, right ventricle hypertrophy, and vascular remodeling in Sugen-5416 combined with chronic hypoxia mice model. Meanwhile, pharmacological inhibition of caspase-11 with wedelolactone exhibited alleviated development of PAH on the monocrotaline-induced rat model. Moreover, knockdown of caspase-4 repressed the onset of TNF-α–induced pyroptosis in human pulmonary arterial endothelial cells and inhibited the activation of pyroptosis effector GSDMD (gasdermin D) and GSDME (gasdermin E). Conclusions: These observations identified the critical role of caspase-4/11 in the pyroptosis pathway to modulate pulmonary vascular dysfunction and accelerate the progression of PAH. Our findings provide a potential diagnostic and therapeutic target in PAH.

Abstract 14698: Matrix Stiffening Induces a Pathogenic Qki-microrna-7-srsf1 Axis in Pulmonary Arterial Endothelial Cells

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Chen-shan J Woodcock ◽  
Neha Hafeez ◽  
Adam Handen ◽  
Ying Tang ◽  
Leonard Estephan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Mrna Level ◽  
Boyden Chamber ◽  
Sequencing Data ◽  
Therapeutic Implications ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Introduction: Early increases in pulmonary artery stiffness drive pathogenic alterations of pulmonary arterial endothelial cells (PAECs), leading to vascular remodeling, a prominent feature of pulmonary hypertension (PH). However, the molecular mechanisms by which PAECs respond to altered extracellular matrix (ECM) mechanics remain unclear. Recent RNA sequencing study of PAECs exposed to stiff versus soft matrices has identified miRNA-7 (miR-7) as a mechanosensitive downregulated miRNA. The biogenesis of miR-7 is inhibited by the RNA binding protein quaking (QKI), which is shown to be correspondingly upregulated in ECM stiffness. Moreover, subsequent RNA sequencing and computational analyses have identified serine and arginine rich splicing factor 1 (SRSF1) as a miR-7 target in PAECs with ECM stiffening. Hypothesis: The QKI-miR-7-SRSF1 axis is a pivotal ECM stiffening-mediated pathogenic signal in PAECs. Methods: Human PAECs were cultured on soft (0.5 or 1 kPa) or stiff (50 kPa) matrices. Lungs from PH patients and rats (SU5416/Hypoxia- and MCT-exposed models) were used to evaluate genes and miRNA expression. Boyden chamber migration analysis examined PAEC mobility. Results: In human and rat PH lungs, miRNA-7 (miR-7) was down-regulated ( P < 0.05). To validate RNA sequencing data by RT-qPCR, QKI level was increased (2.13 ± 0.19, P <0.0001) in PAECs cultured on stiff versus soft matrices and correspondingly miR-7 level was decreased (0.71 ± 0.17, P <0.0001). Furthermore, miR-7 targeted the 3’-untranslated region of SRSF1 mRNA as demonstrated by luciferase activity assay. In PAECs, forced miR-7 overexpression decreased SRSF1, and conversely miR-7 inhibition increased SRSF1. QKI overexpression increased SRSF1 by downregulating miR-7 level. Moreover, forced miR-7 overexpression significantly inhibited PAEC migration ( P <0.0001), accompanied by downregulation of SRSF1 mRNA level (0.75 ± 0.07, P <0.0001). Conclusions: Our data demonstrate that mechanoactivation of a QKI-miR-7-SRSF1 signal axis promotes PAEC pathophenotypes driven by ECM stiffness. These insights broaden our understanding of RNA pathobiology in vascular stiffening and carry important therapeutic implications in PH.

Arginase inhibition increases nitric oxide production in bovine pulmonary arterial endothelial cells

2004 ◽  
Vol 287 (1) ◽  
pp. L60-L68 ◽  
Author(s):  
Louis G. Chicoine ◽  
Michael L. Paffett ◽  
Tamara L. Young ◽  
Leif D. Nelin
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
No Synthase ◽  
No Production ◽  
Urea Production ◽  
Pulmonary Arterial ◽  
Factor Α ◽  
Arterial Endothelial Cells ◽  
Regular Medium ◽  
Necrosis Factor

Nitric oxide (NO) is produced by NO synthase (NOS) from l-arginine (l-Arg). Alternatively, l-Arg can be metabolized by arginase to produce l-ornithine and urea. Arginase (AR) exists in two isoforms, ARI and ARII. We hypothesized that inhibiting AR with l-valine (l-Val) would increase NO production in bovine pulmonary arterial endothelial cells (bPAEC). bPAEC were grown to confluence in either regular medium (EGM; control) or EGM with lipopolysaccharide and tumor necrosis factor-α (L/T) added. Treatment of bPAEC with L/T resulted in greater ARI protein expression and ARII mRNA expression than in control bPAEC. Addition of l-Val to the medium led to a concentration-dependent decrease in urea production and a concentration-dependent increase in NO production in both control and L/T-treated bPAEC. In a second set of experiments, control and L/T bPAEC were grown in EGM, EGM with 30 mM l-Val, EGM with 10 mM l-Arg, or EGM with both 10 mM l-Arg and 30 mM l-Val. In both control and L/T bPAEC, treatment with l-Val decreased urea production and increased NO production. Treatment with l-Arg increased both urea and NO production. The addition of the combination l-Arg and l-Val decreased urea production compared with the addition of l-Arg alone and increased NO production compared with l-Val alone. These data suggest that competition for intracellular l-Arg by AR may be involved in the regulation of NOS activity in control bPAEC and in response to L/T treatment.

scholarly journals Regenerative Effects of Heme Oxygenase Metabolites on Neuroinflammatory Diseases

2018 ◽  
Vol 20 (1) ◽  
pp. 78 ◽  
Author(s):  
Huiju Lee ◽  
Yoon Choi
Keyword(s):  
Endothelial Cells ◽  
Heme Oxygenase ◽  
Neurovascular Unit ◽  
Vascular Diseases ◽  
Cell Types ◽  
Cns Injury ◽  
Perivascular Cells ◽  
Major Barrier ◽  
The Central Nervous System

Heme oxygenase (HO) catabolizes heme to produce HO metabolites, such as carbon monoxide (CO) and bilirubin (BR), which have gained recognition as biological signal transduction effectors. The neurovascular unit refers to a highly evolved network among endothelial cells, pericytes, astrocytes, microglia, neurons, and neural stem cells in the central nervous system (CNS). Proper communication and functional circuitry in these diverse cell types is essential for effective CNS homeostasis. Neuroinflammation is associated with the vascular pathogenesis of many CNS disorders. CNS injury elicits responses from activated glia (e.g., astrocytes, oligodendrocytes, and microglia) and from damaged perivascular cells (e.g., pericytes and endothelial cells). Most brain lesions cause extensive proliferation and growth of existing glial cells around the site of injury, leading to reactions causing glial scarring, which may act as a major barrier to neuronal regrowth in the CNS. In addition, damaged perivascular cells lead to the breakdown of the blood-neural barrier, and an increase in immune activation, activated glia, and neuroinflammation. The present review discusses the regenerative role of HO metabolites, such as CO and BR, in various vascular diseases of the CNS such as stroke, traumatic brain injury, diabetic retinopathy, and Alzheimer’s disease, and the role of several other signaling molecules.

Intracellular redox status affects transplasma membrane electron transport in pulmonary arterial endothelial cells

2002 ◽  
Vol 282 (1) ◽  
pp. L36-L43 ◽  
Author(s):  
Marilyn P. Merker ◽  
Robert D. Bongard ◽  
Nicholas J. Kettenhofen ◽  
Yoshiyuki Okamoto ◽  
Christopher A. Dawson
Keyword(s):  
Endothelial Cells ◽  
Electron Transport ◽  
Control Level ◽  
Redox Status ◽  
Cellular Mechanisms ◽  
Control Activity ◽  
Transplasma Membrane Electron Transport ◽  
Cell Extracts ◽  
Pulmonary Arterial ◽  
Arterial Endothelial Cells

Pulmonary arterial endothelial cells possess transplasma membrane electron transport (TPMET) systems that transfer intracellular reducing equivalents to extracellular electron acceptors. As one aspect of determining cellular mechanisms involved in one such TPMET system in pulmonary arterial endothelial cells in culture, glycolysis was inhibited by treatment with iodoacetate (IOA) or by replacing the glucose in the cell medium with 2-deoxy-d-glucose (2-DG). TPMET activity was measured as the rate of reduction of the extracellular electron acceptor polymer toluidine blue O polyacrylamide. Intracellular concentrations of NADH, NAD+, NADPH, and NADP+ were determined by high-performance liquid chromatography of KOH cell extracts. IOA decreased TPMET activity to 47% of control activity concomitant with a decrease in the NADH/NAD+ ratio to 34% of the control level, without a significant change in the NADPH/NADP+ ratio. 2-DG decreased TPMET activity to 53% of control and decreased both NADH/NAD+ and NADPH/NADP+ ratios to 51% and 55%, respectively, of control levels. When lactate was included in the medium along with the inhibitors, the effects of IOA and 2-DG on both TPMET activity and the NADPH/NADP+ ratios were prevented. The results suggest that cellular redox status is a determinant of pulmonary arterial endothelial cell TPMET activity, with TPMET activity more highly correlated with the poise of the NADH/NAD+redox pair.

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