scholarly journals Endothelial Nitric Oxide Synthase Knockdown in Human Stem Cells Impacts Mitochondrial Biogenesis and Adipogenesis: Live-Cell Real-Time Fluorescence Imaging

2021 ◽  
Vol 10 (4) ◽  
pp. 631
Author(s):  
Sylvia Lee-Huang ◽  
Philip Lin Huang ◽  
Paul Lee Huang
Keyword(s):  
Nitric Oxide ◽  
Stem Cells ◽  
Stem Cell ◽  
Nitric Oxide Synthase ◽  
Real Time ◽  
Mitochondrial Biogenesis ◽  
Endothelial Nitric Oxide Synthase ◽  
Live Cell ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

We carried out live-cell real-time fluorescence imaging to follow the effects of genetic (siRNA) knockdown (KD) of endothelial nitric oxide synthase (eNOS) on mitochondrial biogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). We report here that eNOS KD in hMSCs blocks mitochondrial biogenesis and adipogenesis. The transfer of mitochondria from normal hMSCs to eNOS-deficient hMSCs restores adipogenesis. Furthermore, cell-free mitochondria purified from normal hMSCs also restores adipogenesis in eNOS-deficient cells. Thus, eNOS and NO signaling are essential for mitochondrial biogenesis, and mitochondrial activity is indispensable for adipogenesis in hMSC differentiation. We mapped the path and identified the mechanisms of mitochondrial transfer. We captured real-time images of differentiated mature adipocytes in mitosis and replication. These results reveal that human stem cell-differentiated fat cells are capable of replication. This new finding offers novel insights into our understanding of fat cell expansion and the development of obesity. Real-time imaging in live cells allows synchronized investigation of mitochondrial biogenesis and adipogenesis in stem cell differentiation without reducing living cells to nonliving samples for functional analysis. Live-cell real-time imaging can thus be a faithful and immediate tool for molecular diagnostic medicine. Furthermore, our results suggest that mitochondrial remodeling can be a useful approach in treating adiposity, diabetes, and abnormalities in energy metabolism and vascular signaling.

scholarly journals Mesenchymal stem cells promote mesenteric vasodilation through hydrogen sulfide and endothelial nitric oxide

2019 ◽  
Vol 317 (4) ◽  
pp. G441-G446 ◽  
Author(s):  
Jan Te Winkel ◽  
Quincy E. John ◽  
Brian D. Hosfield ◽  
Natalie A. Drucker ◽  
Amitava Das ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Hydrogen Sulfide ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Wild Type ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Mesenteric ischemia is a devastating process that can result in intestinal necrosis. Mesenchymal stem cells (MSCs) are becoming a promising treatment modality. We hypothesized that 1) MSCs would promote vasodilation of mesenteric arterioles, 2) hydrogen sulfide (H2S) would be a critical paracrine factor of stem cell-mediated vasodilation, 3) mesenteric vasodilation would be impaired in the absence of endothelial nitric oxide synthase (eNOS) within the host tissue, and 4) MSCs would improve the resistin-to-adiponectin ratio in mesenteric vessels. H2S was measured with a specific fluorophore (7-azido-3-methylcoumarin) in intact MSCs and in cells with the H2S-producing enzyme cystathionine β synthase (CBS) knocked down with siRNA. Mechanical responses of isolated second- and third-order mesenteric arteries (MAs) from wild-type and eNOS knockout (eNOSKO) mice were monitored with pressure myography, after which the vessels were snap frozen and later analyzed for resistin and adiponectin via multiplex beaded assay. Addition of MSCs to the myograph bath significantly increased vasodilation of norepinephrine-precontracted MAs. Knockdown of CBS in MSCs decreased H2S production by MSCs and also decreased MSC-initiated MA dilation. MSC-initiated vasodilation was further reduced in eNOSKO vessels. The MA resistin-to-adiponectin ratio was higher in eNOSKO vessels compared with wild-type. These results show that MSC treatment promotes dilation of MAs by an H2S-dependent mechanism. Furthermore, functional eNOS within the host mesenteric bed appears to be essential for maximum stem cell therapeutic benefit, which may be attributable, in part, to modifications in the resistin-to-adiponectin ratio. NEW & NOTEWORTHY Stem cells have been shown to improve survival, mesenteric perfusion, and histological injury scores following intestinal ischemia. These benefits may be due to the paracrine release of hydrogen sulfide. In an ex vivo pressure myography model, we observed that mesenteric arterial dilation improved with stem cell treatment. Hydrogen sulfide release from stem cells and endothelial nitric oxide synthase within the vessels were critical components of optimizing stem cell-mediated mesenteric artery dilation.

scholarly journals Endothelial nitric oxide synthase is dynamically expressed during bone marrow stem cell differentiation into endothelial cells

2007 ◽  
Vol 293 (3) ◽  
pp. H1760-H1765 ◽  
Author(s):  
Zhenguo Liu ◽  
Yuehua Jiang ◽  
Hong Hao ◽  
Kalpna Gupta ◽  
Jian Xu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Growth Factor ◽  
Nitric Oxide Synthase ◽  
Endothelial Nitric Oxide Synthase ◽  
Stem Cell Differentiation ◽  
Enos Expression ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these cells, which was inhibited by NOS inhibitor NG-nitro-l-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.

scholarly journals Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension

2016 ◽  
Vol 310 (1) ◽  
pp. L40-L49 ◽  
Author(s):  
Adeleye J. Afolayan ◽  
Annie Eis ◽  
Maxwell Alexander ◽  
Teresa Michalkiewicz ◽  
Ru-Jeng Teng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Pulmonary Hypertension ◽  
Nitric Oxide Synthase ◽  
Mitochondrial Biogenesis ◽  
Endothelial Nitric Oxide Synthase ◽  
Copy Number ◽  
And Function ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2, partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen.

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