scholarly journals Nitric oxide prevents aortic valve calcification by S-nitrosylation of USP9X to activate NOTCH signaling

2021 ◽  
Vol 7 (6) ◽  
pp. eabe3706
Author(s):  
Uddalak Majumdar ◽  
Sathiyanarayanan Manivannan ◽  
Madhumita Basu ◽  
Yukie Ueyama ◽  
Mark C. Blaser ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Aortic Valve ◽  
Notch Pathway ◽  
Rna Seq ◽  
Calcific Aortic Valve Disease ◽  
Aortic Valves ◽  
Proteomic Approach ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Genetic Deletion

Calcific aortic valve disease (CAVD) is an increasingly prevalent condition, and endothelial dysfunction is implicated in its etiology. We previously identified nitric oxide (NO) as a calcification inhibitor by its activation of NOTCH1, which is genetically linked to human CAVD. Here, we show NO rescues calcification by an S-nitrosylation–mediated mechanism in porcine aortic valve interstitial cells and single-cell RNA-seq demonstrated NO regulates the NOTCH pathway. An unbiased proteomic approach to identify S-nitrosylated proteins in valve cells found enrichment of the ubiquitin-proteasome pathway and implicated S-nitrosylation of USP9X (ubiquitin specific peptidase 9, X-linked) in NOTCH regulation during calcification. Furthermore, S-nitrosylated USP9X was shown to deubiquitinate and stabilize MIB1 for NOTCH1 activation. Consistent with this, genetic deletion of Usp9x in mice demonstrated CAVD and human calcified aortic valves displayed reduced S-nitrosylation of USP9X. These results demonstrate a previously unidentified mechanism by which S-nitrosylation–dependent regulation of a ubiquitin-associated pathway prevents CAVD.

scholarly journals Nitric oxide prevents aortic valve calcification by S-nitrosylation of USP9X to activate NOTCH signaling

2020 ◽  
Author(s):  
Uddalak Majumdar ◽  
Sathiyanarayanan Manivannan ◽  
Madhumita Basu ◽  
Yukie Ueyama ◽  
Mark C. Blaser ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Aortic Valve ◽  
Aortic Valve Disease ◽  
Notch Pathway ◽  
Valve Disease ◽  
Rna Seq ◽  
Calcific Aortic Valve Disease ◽  
Proteomic Approach ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

AbstractCalcific aortic valve disease (CAVD) is an increasingly prevalent condition and endothelial dysfunction is implicated in its etiology. We previously identified nitric oxide (NO) as a calcification inhibitor by its activation of NOTCH1, which is genetically linked to human CAVD. Here, we show that NO rescues calcification by a S-nitrosylation-mediated mechanism in porcine aortic valve interstitial cells (pAVICs) and single cell RNA-seq demonstrated regulation of NOTCH pathway by NO. A unbiased proteomic approach to identify S-nitrosylated proteins in valve cells found enrichment of the ubiquitin proteasome pathway and implicated S-nitrosylation of USP9X in NOTCH regulation during calcification. Furthermore, S-nitrosylated USP9X was shown to deubiquitinate and stabilize MIB1 for NOTCH1 activation. Consistent with this, genetic deletion of Usp9x in mice demonstrated aortic valve disease and human calcified aortic valves displayed reduced S-nitrosylation of USP9X. These results demonstrate a novel mechanism by which S-nitrosylation dependent regulation of ubiquitin-associated pathway prevents CAVD.

scholarly journals Metabolomics in Severe Aortic Stenosis Reveals Intermediates of Nitric Oxide Synthesis as Most Distinctive Markers

2021 ◽  
Vol 22 (7) ◽  
pp. 3569
Author(s):  
Beau Olivier van van Driel ◽  
Maike Schuldt ◽  
Sila Algül ◽  
Evgeni Levin ◽  
Ahmet Güclü ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cardiac Surgery ◽  
Aortic Valve ◽  
Aortic Stenosis ◽  
Western Blot ◽  
Aortic Valve Disease ◽  
Metabolic Profile ◽  
Valve Disease ◽  
Healthy Controls ◽  
Calcific Aortic Valve Disease

Background: Calcific aortic valve disease (CAVD) is a rapidly growing global health problem with an estimated 12.6 million cases globally in 2017 and a 112% increase of deaths since 1990 due to aging and population growth. CAVD may develop into aortic stenosis (AS) by progressive narrowing of the aortic valve. AS is underdiagnosed, and if treatment by aortic valve replacement (AVR) is delayed, this leads to poor recovery of cardiac function, absence of symptomatic improvement and marked increase of mortality. Considering the current limitations to define the stage of AS-induced cardiac remodeling, there is need for a novel method to aid in the diagnosis of AS and timing of intervention, which may be found in metabolomics profiling of patients. Methods: Serum samples of nine healthy controls and 10 AS patients before and after AVR were analyzed by untargeted mass spectrometry. Multivariate modeling was performed to determine a metabolic profile of 30 serum metabolites which distinguishes AS patients from controls. Human cardiac microvascular endothelial cells (CMECs) were incubated with serum of the AS patients and then stained for ICAM-1 with Western Blot to analyze the effect of AS patient serum on endothelial cell activation. Results: The top 30 metabolic profile strongly distinguishes AS patients from healthy controls and includes 17 metabolites related to nitric oxide metabolism and 12 metabolites related to inflammation, in line with the known pathomechanism for calcific aortic valve disease. Nine metabolites correlate strongly with left ventricular mass, of which three show reversal back to control values after AVR. Western blot analysis of CMECs incubated with AS patient sera shows a significant reduction (14%) in ICAM-1 in AS samples taken after AVR compared to AS patient sera before AVR. Conclusion: Our study defined a top 30 metabolic profile with biological and clinical relevance, which may be used as blood biomarker to identify AS patients in need of cardiac surgery. Future studies are warranted in patients with mild-to-moderate AS to determine if these metabolites reflect disease severity and can be used to identify AS patients in need of cardiac surgery.

scholarly journals Interleukin-37 suppresses the osteogenic responses of human aortic valve interstitial cells in vitro and alleviates valve lesions in mice

2017 ◽  
Vol 114 (7) ◽  
pp. 1631-1636 ◽  
Author(s):  
Qingchun Zeng ◽  
Rui Song ◽  
David A. Fullerton ◽  
Lihua Ao ◽  
Yufeng Zhai ◽  
...  
Keyword(s):  
Aortic Valve ◽  
High Fat Diet ◽  
Aortic Valve Disease ◽  
Valve Disease ◽  
High Fat ◽  
Calcific Aortic Valve Disease ◽  
Aortic Valves ◽  
Valve Interstitial Cells ◽  
Valve Lesions ◽  
Osteogenic Factors

Calcific aortic valve disease is a chronic inflammatory process, and aortic valve interstitial cells (AVICs) from diseased aortic valves express greater levels of osteogenic factors in response to proinflammatory stimulation. Here, we report that lower cellular levels of IL-37 in AVICs of diseased human aortic valves likely account for augmented expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP) following stimulation of Toll-like receptor (TLR) 2 or 4. Treatment of diseased AVICs with recombinant human IL-37 suppresses the levels of BMP-2 and ALP as well as calcium deposit formation. In mice, aortic valve thickening is observed when exposed to a TLR4 agonist or a high fat diet for a prolonged period; however, mice expressing human IL-37 exhibit significantly lower BMP-2 levels and less aortic valve thickening when subjected to the same regimens. A high fat diet in mice results in oxidized low-density lipoprotein (oxLDL) deposition in aortic valve leaflets. Moreover, the osteogenic responses in human AVICs induced by oxLDL are suppressed by recombinant IL-37. Mechanistically, reduced osteogenic responses to oxLDL in human AVICs are associated with the ability of IL-37 to inhibit NF-κB and ERK1/2. These findings suggest that augmented expression of osteogenic factors in AVICs of diseased aortic valves from humans is at least partly due to a relative IL-37 deficiency. Because recombinant IL-37 suppresses the osteogenic responses in human AVICs and alleviates aortic valve lesions in mice exposed to high fat diet or a proinflammatory stimulus, IL-37 has therapeutic potential for progressive calcific aortic valve disease.

scholarly journals COX-2 Is Downregulated in Human Stenotic Aortic Valves and Its Inhibition Promotes Dystrophic Calcification

2020 ◽  
Vol 21 (23) ◽  
pp. 8917
Author(s):  
Francesco Vieceli Dalla Sega ◽  
Francesca Fortini ◽  
Paolo Cimaglia ◽  
Luisa Marracino ◽  
Elisabetta Tonet ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Clinical Analysis ◽  
Dystrophic Calcification ◽  
Calcific Aortic Valve Disease ◽  
Aortic Valves ◽  
Inflammatory Drugs ◽  
Cox 2 ◽  
Anti Inflammatory ◽  
Induced Apoptosis ◽  
Human Aortic Valve

Calcific aortic valve disease (CAVD) is the result of maladaptive fibrocalcific processes leading to a progressive thickening and stiffening of aortic valve (AV) leaflets. CAVD is the most common cause of aortic stenosis (AS). At present, there is no effective pharmacotherapy in reducing CAVD progression; when CAVD becomes symptomatic it can only be treated with valve replacement. Inflammation has a key role in AV pathological remodeling; hence, anti-inflammatory therapy has been proposed as a strategy to prevent CAVD. Cyclooxygenase 2 (COX-2) is a key mediator of the inflammation and it is the target of widely used anti-inflammatory drugs. COX-2-inhibitor celecoxib was initially shown to reduce AV calcification in a murine model. However, in contrast to these findings, a recent retrospective clinical analysis found an association between AS and celecoxib use. In the present study, we investigated whether variations in COX-2 expression levels in human AVs may be linked to CAVD. We extracted total RNA from surgically explanted AVs from patients without CAVD or with CAVD. We found that COX-2 mRNA was higher in non-calcific AVs compared to calcific AVs (0.013 ± 0.002 vs. 0.006 ± 0.0004; p < 0.0001). Moreover, we isolated human aortic valve interstitial cells (AVICs) from AVs and found that COX-2 expression is decreased in AVICs from calcific valves compared to AVICs from non-calcific AVs. Furthermore, we observed that COX-2 inhibition with celecoxib induces AVICs trans-differentiation towards a myofibroblast phenotype, and increases the levels of TGF-β-induced apoptosis, both processes able to promote the formation of calcific nodules. We conclude that reduced COX-2 expression is a characteristic of human AVICs prone to calcification and that COX-2 inhibition may promote aortic valve calcification. Our findings support the notion that celecoxib may facilitate CAVD progression.

scholarly journals TLR4 Stimulation Promotes Human AVIC Fibrogenic Activity through Upregulation of Neurotrophin 3 Production

2020 ◽  
Vol 21 (4) ◽  
pp. 1276
Author(s):  
Qingzhou Yao ◽  
Erlinda The ◽  
Lihua Ao ◽  
Yufeng Zhai ◽  
Maren K. Osterholt ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Critical Role ◽  
Chronic Inflammatory Disease ◽  
Collagen Deposition ◽  
Calcific Aortic Valve Disease ◽  
Aortic Valves ◽  
Neurotrophin 3 ◽  
Collagen Iii ◽  
Inflammatory Stimuli ◽  
Stimulation Of

Background: Calcific aortic valve disease (CAVD) is a chronic inflammatory disease that manifests as progressive valvular fibrosis and calcification. An inflammatory milieu in valvular tissue promotes fibrosis and calcification. Aortic valve interstitial cell (AVIC) proliferation and the over-production of the extracellular matrix (ECM) proteins contribute to valvular thickening. However, the mechanism underlying elevated AVIC fibrogenic activity remains unclear. Recently, we observed that AVICs from diseased aortic valves express higher levels of neurotrophin 3 (NT3) and that NT3 exerts pro-osteogenic and pro-fibrogenic effects on human AVICs. Hypothesis: Pro-inflammatory stimuli upregulate NT3 production in AVICs to promote fibrogenic activity in human aortic valves. Methods and Results: AVICs were isolated from normal human aortic valves and were treated with lipopolysaccharide (LPS, 0.20 µg/mL). LPS induced TLR4-dependent NT3 production. This effect of LPS was abolished by inhibition of the Akt and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways. The stimulation of TLR4 in human AVICs with LPS resulted in a greater proliferation rate and an upregulated production of matrix metallopeptidases-9 (MMP-9) and collagen III, as well as augmented collagen deposition. Recombinant NT3 promoted AVIC proliferation in a tropomyosin receptor kinase (Trk)-dependent fashion. The neutralization of NT3 or the inhibition of Trk suppressed LPS-induced AVIC fibrogenic activity. Conclusions: The stimulation of TLR4 in human AVICs upregulates NT3 expression and promotes cell proliferation and collagen deposition. The NT3-Trk cascade plays a critical role in the TLR4-mediated elevation of fibrogenic activity in human AVICs. Upregulated NT3 production by endogenous TLR4 activators may contribute to aortic valve fibrosis associated with CAVD progression.

scholarly journals Inhibition of the Ubiquitin—Proteasome Pathway Alters Cellular Levels of Nitric Oxide in Tomato Seedlings

2010 ◽  
Vol 3 (5) ◽  
pp. 854-869 ◽  
Author(s):  
Sangeeta Negi ◽  
Parankusam Santisree ◽  
Eros Vasil Kharshiing ◽  
Rameshwar Sharma
Keyword(s):  
Nitric Oxide ◽  
Ubiquitin Proteasome Pathway ◽  
Tomato Seedlings ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway

scholarly journals Interstitial cells in calcified aortic valves have reduced differentiation potential and stem cell-like properties

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maria Bogdanova ◽  
Arsenii Zabirnyk ◽  
Anna Malashicheva ◽  
Katarina Zihlavnikova Enayati ◽  
Tommy Aleksander Karlsen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Aortic Valve ◽  
Collagen Gel ◽  
Interstitial Cells ◽  
Osteogenic Medium ◽  
Calcific Aortic Valve Disease ◽  
Aortic Valves ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Lower Expression

Abstract Valve interstitial cells (VICs) are crucial in the development of calcific aortic valve disease. The purpose of the present investigation was to compare the phenotype, differentiation potential and stem cell-like properties of cells from calcified and healthy aortic valves. VICs were isolated from human healthy and calcified aortic valves. Calcification was induced with osteogenic medium. Unlike VICs from healthy valves, VICs from calcified valves cultured without osteogenic medium stained positively for calcium deposits with Alizarin Red confirming their calcific phenotype. Stimulation of VICs from calcified valves with osteogenic medium increased calcification (p = 0.02), but not significantly different from healthy VICs. When stimulated with myofibroblastic medium, VICs from calcified valves had lower expression of myofibroblastic markers, measured by flow cytometry and RT-qPCR, compared to healthy VICs. Contraction of collagen gel (a measure of myofibroblastic activity) was attenuated in cells from calcified valves (p = 0.04). Moreover, VICs from calcified valves, unlike cells from healthy valves had lower potential to differentiate into adipogenic pathway and lower expression of stem cell-associated markers CD106 (p = 0.04) and aldehyde dehydrogenase (p = 0.04). In conclusion, VICs from calcified aortic have reduced multipotency compared to cells from healthy valves, which should be considered when investigating possible medical treatments of aortic valve calcification.

scholarly journals A Proteomic View of Cellular Responses to Anticancer Quinoline-Copper Complexes

Proteomes ◽  
2019 ◽  
Vol 7 (2) ◽  
pp. 26 ◽  
Author(s):  
Bastien Dalzon ◽  
Joanna Bons ◽  
Hélène Diemer ◽  
Véronique Collin-Faure ◽  
Caroline Marie-Desvergne ◽  
...  
Keyword(s):  
Copper Complex ◽  
Copper Complexes ◽  
Leukemic Cell ◽  
Anticancer Activities ◽  
Proteomic Approach ◽  
Target Action ◽  
Biological Target ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
Validation Experiments

Metal-containing drugs have long been used in anticancer therapies. The mechansims of action of platinum-based drugs are now well-understood, which cannot be said of drugs containing other metals, such as gold or copper. To gain further insights into such mechanisms, we used a classical proteomic approach based on two-dimensional elelctrophoresis to investigate the mechanisms of action of a hydroxyquinoline-copper complex, which shows promising anticancer activities, using the leukemic cell line RAW264.7 as the biological target. Pathway analysis of the modulated proteins highlighted changes in the ubiquitin/proteasome pathway, the mitochondrion, the cell adhesion-cytoskeleton pathway, and carbon metabolism or oxido-reduction. In line with these prteomic-derived hypotheses, targeted validation experiments showed that the hydroxyquinoline-copper complex induces a massive reduction in free glutathione and a strong alteration in the actin cytoskeleton, suggesting a multi-target action of the hydroxyquinoline-copper complex on cancer cells.

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