scholarly journals POS0041 THE GASOTRANSMITTER HYDROGEN SULFIDE (H2S) IS PROTECTIVE AGAINST CALCIFIC TENDINOPATHY (CT)

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 226.2-226
Author(s):  
I. Bernabei ◽  
D. Kronenberg ◽  
R. Stange ◽  
J. Bertrand ◽  
T. Hügle ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Rotator Cuff ◽  
Achilles Tendon ◽  
Collagen Crosslinks ◽  
Painful Condition ◽  
Calcific Tendinopathy ◽  
Expression And Activity

Background:Pathological (or heterotopic) calcification is the deposition of calcium-containing crystals in soft tissues that normally do not calcify. The deposition of these crystals in tendons such as the rotator cuff and the Achilles tendon is known as calcific tendinopathy (CT). CT is a painful condition, which increases tendon rupture rate and leads to disability.Objectives:To understand what inhibits calcification, in order to provide new strategies to treat a condition for which existing therapies are ineffective.Methods:We investigated the role of the gasotransmitter hydrogen sulfide (H2S), and in particular of the H2S-producing enzyme cystathionine γ-lyase (CSE) in CT. In vitro, we induced calcification in tenocytes from WT and CSE KO mice or we treated WT tenocytes with different H2S donors. In vivo, calcification was assessed in a surgery-induced murine model of CT (tenotomy of the Achilles tendon) and in a spontaneous model of CT (aging). Samples obtained from patients with rotator cuff or Achilles tendon CT were also analyzed. To investigate the underlying mechanisms of the CSE-H2S effect, we focused on the bone morphogenic proteins (BMPs) pathway. We additionally explored if altered extracellular matrix (ECM) organization, due to lysyl oxidase (LOX) activity and aberrant collagen-crosslinks, could also be involved in CT. In this context, we studied if H2S could affect LOX expression and activity.Results:In vitro, tenocyte calcification was inhibited by exogenous H2S-donors, while it was exacerbated in CSE KO tenocytes. The protective role of CSE-H2S was confirmed in vivo. In aged mice, microtomography analysis revealed exacerbated Achilles tendon calcification in CSE KO mice compared to WT. In the surgery-induced model of CT, an inverse correlation between calcification and CSE expression in operated Achilles tendon was seen over time. Similarly, inversed correlation between calcification and CSE expression was found in human CT samples.Reduced calcification in tenocytes exposed to H2S was accompanied by decreased expression of genes coding for BMP2, BMP4 and decreased activation of the BMP signaling pathway (pSMAD1/5/8). On the contrary, BMPs expression and BMPs-pathway activation were exacerbated in CSE KO tenocytes compared to WT tenocytes.We next investigated whether ECM disorganization could play a role in CT. Tenocytes cultured in calcification media and treated with the pan-inhibitor of lysyl oxidases (LOX, LOXL1-4) β-aminopropionitrile (BAPN) showed decreased calcification. This pointed to a potential beneficial role of LOX inhibition, therefore decreased collagen-crosslinks, in CT. By analysis of LOXs gene expression in WT and CSE KO tenocytes cultured in calcifying condition, we found much higher expression (4-fold) of LOX, LOL2 and LOXL4 in CSE KO tenocytes. Moreover, H2S-donors inhibited LOX activity. Altogether, these results suggest that decreased H2S could lead to aberrant LOX expression and activity, excessive collagen cross-links in the ECM, and ultimately calcification. Further experiments are ongoing to prove these hypotheses.Conclusion:We suggest targeting H2S production by CSE, or supplying an H2S-donor, is of therapeutic relevance to pathological calcification in the context of CT and can modify its disease course.The anti-mineralizing effect of H2S in tendons could be due to both inhibition of the BMPs pathway and suppression of abnormal LOXs activity.Disclosure of Interests:None declared.

scholarly journals Basal Polarity Complex Scribble Is Required for Leukemic Initiation and Propagation through Negative Regulation of Apical Polarity Complex Activator Cdc42 and Hypoxia Inducing Factor-1α

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 551-551
Author(s):  
Shailaja N Hegde ◽  
Mark J Althoff ◽  
Ramesh C. Nayak ◽  
Ashley M Wellendorf ◽  
Fatima Mohmoud ◽  
...  
Keyword(s):  
Median Survival ◽  
Leukemic Cells ◽  
Mtorc1 Signaling ◽  
Apical Polarity ◽  
B Lymphoid ◽  
Deficient Mice ◽  
Expression And Activity

Abstract Despite the introduction of tyrosine kinase inhibitor and CAR-T cell therapies, the prognosis for Ph+ and Ph-like acute lymphoblastic leukemia remains poor. In the present study, we show the role of the Scribble protein in both lymphoid and myeloid leukemogenesis. The polarity protein Scribble is a member of the basal polarity complex, which is down-regulated in many cancers, suggesting a possible tumor suppressor role, especially in so-called cancer initiating cells. Its effect and mechanisms of activity in leukemic cell fate along with its potential activity on leukemic initiating cells have only been recently started to elucidated. Using interferon-responsive inducible (Mx1-Cre) Scribble-deficient mice, we have characterized the role of Scribble in both retroviral transduction, transplantation animal models and binary, inducible stem cell initiated (Scl-tTA/TRE-BCR-ABL) serial propagation models of BCR-ABL induced leukemia. We found that Scribble expression is upregulated at both transcriptional and translational levels in p210- or p190-BCR-ABL induced leukemic progenitors. In vitro, leukemic colony formation was impaired in Scribble deficient leukemic progenitors (~48% reduction; p≤ 0.05, compared to Wt leukemic progenitors) demonstrating that Scribble is important for leukemogenesis. In vivo, the deletion of Scribble abrogates the development of myeloproliferative disease induced by p210-BCR-ABL (median survival: 70 vs 47 days in Scribble deficient and Wt chimeric mice, respectively; p≤0.05); and significantly impairs B-cell lymphoid leukemogenesis induced by p190-BCR-ABL (median survival: 80 vs 60 days for Scribble deficient and Wt chimeric animals, respectively). Mechanistically, BCR-ABL activates the apical polarity regulator Cdc42 in leukemic progenitors and this activation is inhibited by the deficiency of Scribble. The deficiency of Cdc42 does not impair leukemogenesis but the combined deficiency of Cdc42 and Scribble restores the in vivo survival (median survival: 47 days, p≤0.01 compared to Scribble deficient mice) in chimeric p190-BCR-ABL+ leukemic mice to levels similar to wild-type leukemic cells. These data indicate that Scribble-deficient leukemogenesis is dependent on oncogene induced Cdc42 activity in lymphoid progenitors. Furthermore, Scribble deficiency in leukemic progenitors increases the activation of the AMPK/mTORC1 signaling pathway and the protein expression and transcriptional activity of its downstream effector hypoxia-inducing factor-1α (HIF-1α). HIF-1α silencing by constitutive shRNA expression or inducible deletion in Scribble deleted B-lymphoid leukemic cells restored leukemic progenitor clonogenic efficiency (CFU average: 52 vs 110 per 1,000 B220+/EGFP+ BM cells, in Scribble and double Scribble/HIF-1α deficient, respectively; p≤0.01) and B-lymphoid leukemogenesis in vivo (median survival of 62 days; p≤0.05 compared with Scribble deficient chimeric animals). In addition, double deficiency of Scribble and HIF-1α restored AMPK/mTORC1 signaling to Wt leukemic levels. This data indicates that Scribble is a negative regulator of HIF-1α expression and activity, and the restoration of HIF-1α expression and activity to normal leukemic levels is necessary to restore leukemogenesis. Altogether, our data indicates that Scribble is a positive regulator of oncogenesis in leukemic progenitors, in vitro and in vivo, through Cdc42 and HIF-1α activities. Disclosures No relevant conflicts of interest to declare.

scholarly journals 2027 The role of lysyl oxidase in systemic sclerosis-associated lung fibrosis

2018 ◽  
Vol 2 (S1) ◽  
pp. 32-33
Author(s):  
Xinh-Xinh Nguyen ◽  
Tetsuya Nishimoto ◽  
Takahisa Takihara ◽  
Logan Mlakar ◽  
Ellen Riemer ◽  
...  
Keyword(s):  
Lung Fibrosis ◽  
Human Lung ◽  
Ex Vivo ◽  
Lysyl Oxidase ◽  
Lung Fibroblasts ◽  
Primary Fibroblasts ◽  
Expression And Activity

OBJECTIVES/SPECIFIC AIMS: Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology characterized by progressive fibrosis of the skin and multiple visceral organs. Effective therapies for SSc are needed. Lysyl oxidase (LOX) is a copper-dependent amide oxidase that plays a critical role in the crosslinking of the extracellular matrix (ECM). In this study, we investigated the role of LOX in the pathophysiology of SSc. METHODS/STUDY POPULATION: LOX expression and protein levels were measured in lung tissues and primary fibroblasts from patients with SSc and healthy controls. The effects of recombinant LOX (rLOX) were measured in vitro in primary fibroblasts, ex vivo in human lung tissues and in vivo in mice given bleomycin in combination with rLOX. LOX levels and activity were evaluated in lung fibroblasts treated with an endostatin-derived peptide that ameliorates fibrosis and in mice treated with bleomycin in combination with the peptide. Further, to differentiate the crosslinking activity of LOX from other potential effects, primary human fibroblasts were cultured with rLOX in the presence of the inhibitor, beta-aminopropionitrile. The expression levels of ECM (collagen and fibronectin), pro-fibrotic factors (IL-6 and TGF-beta), and transcription factor (c-Fos) were examined by real-time PCR, ELISA, immunoblotting, or hydroxyproline assay. RESULTS/ANTICIPATED RESULTS: LOX mRNA was increased in lung tissues and matching fibroblasts of SSc patients. rLOX-induced ECM production in vitro and ex vivo in lung fibroblasts and in human lung tissues maintained in organ culture, respectively. Additionally, TGF-beta and bleomycin induced ECM production, LOX mRNA expression and activity. Endostatin peptide abrogated these effects. In vivo, rLOX synergistically exacerbated pulmonary fibrosis in bleomycin-treated mice. The inhibition of LOX catalytic activity by beta-aminopropionitrile failed to abrogate LOX-induced ECM production. LOX increased the production of IL-6. IL-6 neutralization blocked the effects of LOX. Further, LOX induced c-Fos expression and its nuclear localization. DISCUSSION/SIGNIFICANCE OF IMPACT: LOX expression and activity were increased with fibrosis in vitro, ex vivo, and in vivo. LOX induced fibrosis via increasing ECM, IL-6 and c-Fos translocation to the nucleus. These effects were independent of the crosslinking activity of LOX and mediated by IL-6. Our findings suggest that inhibition of LOX may be a viable option for the treatment of lung fibrosis. Further, the use of human lung in organ culture establishes the relevance of our findings to human disease.

scholarly journals Synergisms, Discrepancies and Interactions between Hydrogen Sulfide and Carbon Monoxide in the Gastrointestinal and Digestive System Physiology, Pathophysiology and Pharmacology

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 445 ◽  
Author(s):  
Urszula Głowacka ◽  
Tomasz Brzozowski ◽  
Marcin Magierowski
Keyword(s):  
Carbon Monoxide ◽  
Hydrogen Sulfide ◽  
Digestive System ◽  
In Vitro Models ◽  
Biological Functions ◽  
Systems Physiology ◽  
System Physiology

Endogenous gas transmitters, hydrogen sulfide (H2S), carbon monoxide (CO) and nitric oxide (NO) are important signaling molecules known to exert multiple biological functions. In recent years, the role of H2S, CO and NO in regulation of cardiovascular, neuronal and digestive systems physiology and pathophysiology has been emphasized. Possible link between these gaseous mediators and multiple diseases as well as potential therapeutic applications has attracted great attention from biomedical scientists working in many fields of biomedicine. Thus, various pharmacological tools with ability to release CO or H2S were developed and implemented in experimental animal in vivo and in vitro models of many disorders and preliminary human studies. This review was designed to review signaling functions, similarities, dissimilarities and a possible cross-talk between H2S and CO produced endogenously or released from chemical donors, with special emphasis on gastrointestinal digestive system pathologies prevention and treatment.

scholarly journals Hydrogen Sulfide as a Potential Therapeutic Target in Fibrosis

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Shufang Zhang ◽  
Chuli Pan ◽  
Feifei Zhou ◽  
Zhi Yuan ◽  
Huiying Wang ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Protective Role ◽  
Potential Therapeutic Target ◽  
Future Studies ◽  
Abnormal Metabolism ◽  
And Function ◽  
Anti Inflammation

Hydrogen sulfide (H2S), produced endogenously by the activation of two major H2S-generating enzymes (cystathionineβ-synthase and cystathionineγ-lyase), plays important regulatory roles in different physiologic and pathologic conditions. The abnormal metabolism of H2S is associated with fibrosis pathogenesis, causing damage in structure and function of different organs. A number ofin vivoandin vitrostudies have shown that both endogenous H2S level and the expressions of H2S-generating enzymes in plasma and tissues are significantly downregulated during fibrosis. Supplement with exogenous H2S mitigates the severity of fibrosis in various experimental animal models. The protective role of H2S in the development of fibrosis is primarily attributed to its antioxidation, antiapoptosis, anti-inflammation, proangiogenesis, and inhibition of fibroblasts activities. Future studies might focus on the potential to intervene fibrosis by targeting the pathway of endogenous H2S-producing enzymes and H2S itself.

Abstract 18: The Role of Matrix Metalloproteinase 12 in Vascular Stiffness, Inflammation and Hypertension

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Nataliya A Pidkovka ◽  
Jing Wu ◽  
Anna E Goldstein ◽  
Salim Thabet ◽  
David G Harrison
Keyword(s):  
Endothelial Cells ◽  
Angiotensin Ii ◽  
Mechanical Stretch ◽  
Pressure Change ◽  
Vascular Stiffness ◽  
Western Blots ◽  
Expression And Activity

Vascular wall remodeling and inflammation contribute to hypertension. These processes decrease arterial elasticity due to a loss of elastin and deposition of collagen. Metalloproteinase-12 (MMP12) is an elastase produced by macrophages and vascular cells. Cleavage of elastin by MMP12 could increase vascular stiffness and lead to release of pro-inflammatory elastin fragments. We sought to determine if MMP12 contributes to hypertension and vascular stiffness in response to angiotensin II and to understand mechanisms responsible for its expression. To determine if mechanical stretch activates MMP12 we subjected cultured murine endothelial cells to 0%, 5% (normotensive) or 10% (hypertensive) uniaxial stretch for 48 hours. Real-time RT-PCR showed that 10% stretch increases MMP12 mRNA by 4 fold compared to 0% or 5% stretch. Western blots and casein zymography indicated that 10% stretch significantly increases MMP12 protein expression and activity, respectively. Western blots also revealed an increase in elastin fragments in the media of cells exposed to 10% stretch compared to those exposed to 5% stretch. To understand the role of MMP12 in hypertension, we infused angiotensin II in wild-type and MMP12 -/- mice. In keeping with the studies of cultured endothelial cells, angiotensin II markedly increased vascular MMP12 mRNA (10-fold) and protein levels. The hypertension caused by angiotensin II was markedly blunted in MMP12 -/- mice (122 ± 3 vs. 173 ± 5, p < 0.0001). Immunohistochemistry with a CD68 specific antibody indicated a significant decrease in macrophages accumulation in the perivascular tissues of MMP12 -/- compared to WT mice. We also examined distensibility of aortic segments in vitro, and quantified this as the percent change in diameter over a pressure change from 75 to 125 mmHg. Angiotensin II markedly decreased this distensibility index from 24 ± 3 to 9 ± 2%, but only to 16 ± 3% in MMP12 -/- mice (p < 0.05). In conclusion, these studies show that mechanical stretch in vitro and hypertension in vivo cause a striking increase in endothelial cell and vascular MMP12 expression and activity. MMP12 activation promotes aortic stiffening and ultimately hypertension. Thus, modulation of MMP12 might be a therapeutic target in hypertension.

Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation

2019 ◽  
Vol 133 (20) ◽  
pp. 2045-2059 ◽  
Author(s):  
Da Zhang ◽  
Xiuli Wang ◽  
Siyao Chen ◽  
Selena Chen ◽  
Wen Yu ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Cell Model ◽  
Site Directed Mutagenesis ◽  
Critical Event ◽  
Hypertensive Rats ◽  
Pulmonary Artery Endothelial Cell

Abstract Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Anti-obesity role of Aster glehni extract: in vivo and in vitro effects

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
HM Lee ◽  
TG Ahn ◽  
CW Kim ◽  
HJ An
Keyword(s):  
In Vitro Effects

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

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