The Gasotransmitter Hydrogen Sulfide (H2S) Prevents Pathologic Calcification (PC) in Cartilage.

Pathologic calcification (PC) is a painful and disabling condition whereby calcium-containing crystals deposit in tissues that do not physiologically calcify: cartilage, tendons, muscle, vessels and skin. In cartilage, compression and inflammation triggered by PC leads to cartilage degradation typical of osteoarthritis (OA). The PC process is poorly understood and treatments able to target the underlying mechanisms of the disease are lacking. Here we show a crucial role of the gasotransmitter hydrogen sulfide (H2S) and, in particular, of the H2S-producing enzyme cystathionine γ-lyase (CSE), in regulating PC in cartilage. Cse deficiency (Cse KO mice) exacerbated calcification in both surgically-induced (menisectomy) and spontaneous (aging) murine models of cartilage PC, and augmented PC was closely associated with cartilage degradation (OA). On the contrary, Cse overexpression (Cse tg mice) protected from these features. In vitro, Cse KO chondrocytes showed increased calcification, potentially via enhanced alkaline phosphatase (Alpl) expression and activity and increased IL-6 production. The opposite results were obtained in Cse tg chondrocytes. In cartilage samples from patients with OA, CSE expression inversely correlated with the degree of tissue calcification and disease severity. Increased cartilage degradation in murine and human tissues lacking or expressing low CSE levels may be accounted for by dysregulated catabolism. We found higher levels of matrix-degrading metalloproteases Mmp-3 and -13 in Cse KO chondrocytes, whereas the opposite results were obtained in Cse tg cells. Finally, by high-throughput screening, we identified a novel small molecule CSE positive allosteric modulator (PAM), and demonstrated that it was able to increase cellular H2S production, and decrease murine and human chondrocyte calcification and IL-6 secretion. Together, these data implicate impaired CSE-dependent H2S production by chondrocytes in the etiology of cartilage PC and worsening of secondary outcomes (OA). In this context, enhancing CSE expression and/or activity in chondrocytes could represent a potential strategy to inhibit PC.

Xanthine Oxidoreductase Is Involved in Chondrocyte Mineralization and Expressed in Osteoarthritic Damaged Cartilage.

Pathologic calcification of cartilage consists of the formation of basic calcium phosphate (BCP) and/or calcium pyrophosphate dihydrate (CPPD) containing calcium crystals in mature hyaline or articular cartilage and is associated with aging, cartilage injury and likely plays a role in accelerating the pathology of osteoarthritis (OA). The pathways regulating joint calcification, in particular cartilage calcification, are not completely understood, but inflammation and the formation of reactive oxygen species (ROS) are contributory factors. The xanthine oxidase (XO) form of xanthine oxidoreductase (XOR), the key enzyme in xanthine and uric acid metabolism, is a major cellular source of superoxide. We hypothesized that XOR could be implicated in chondrocyte mineralization and cartilage calcification and degradation in OA. We showed both in murine primary chondrocyte and chondrogenic ATDC5 cells, that mineralization was inhibited by two different XOR inhibitors, febuxostat and allopurinol. In addition, XOR inhibition reduced the expression of the pro-mineralizing cytokine interleukin-6 (IL-6). We next generated XOR knock-out chondrocyte cell lines with undetectable XOR expression and XO activity. XOR knock-out chondrocyte cells showed decreased mineralization and reduced alkaline phosphatase (Alp) activity. To assess the precise form of XOR involved, primary chondrocytes of XOR mutant mice expressing either the XDH form (XDH ki) or the XO form (XO ki) were studied. We found that XO ki chondrocytes exhibited increased mineralization compared to XDH ki chondrocytes, and this was associated with enhanced Alp activity, ROS generation and IL-6 secretion. Finally, we found increased XOR expression in damaged vs. undamaged cartilage obtained from OA patients and XOR expression partially co-localized with areas showing pathologic calcification. Altogether, our results suggest that XOR, via its XO form, contribute to chondrocyte mineralization and pathological calcification in OA cartilage.

CD11b Signaling Prevents Chondrocyte Mineralization and Attenuates the Severity of Osteoarthritis.

Osteoarthritis (OA) is a progressive joint disease that is strongly associated with calcium-containing crystal formation (mineralization) by chondrocytes leading ultimately to cartilage calcification. However, this calcification process is poorly understood and treatments targeting the underlying disease mechanisms are lacking. The CD11b/CD18 integrin (Mac-1 or αMß2), a member of the beta 2 integrin family of adhesion receptors, is critically involved in the development of several inflammatory diseases, including rheumatoid arthritis and systemic lupus erythematosus. We found that in a collagen-induced arthritis, CD11b-deficient mice exhibited increased cartilage degradation compared to WT control animals. However, the functional significance of CD11b integrin signaling in the pathophysiology of chondrocytes remains unknown. CD11b expression was found in the extracellular matrix and in chondrocytes in both healthy and damaged human and murine articular cartilage. Primary murine CD11b KO chondrocytes showed increased mineralization when induced in vitro by secondary calciprotein particles (CPP) and quantified by Alizarin Red staining. This increased propensity to mineralize was associated with an increased alkaline phosphatase (Alp) expression (measured by qRT-PCR and activity assay) and an enhanced secretion of the pro-mineralizing IL-6 cytokine compared to control wild-type cells (measured by ELISA). Accordingly, addition of an anti-IL-6 receptor antibody to CD11b KO chondrocytes reduced significantly the calcification and identified IL-6 as a pro-mineralizing factor in these cells. In the same conditions, the ratio of qRT-PCR expression of collagen X over collagen II, and that of Runx2 over Sox9 (both ratio being indexes of chondrocyte hypertrophy) were increased in CD11b-deficient cells. Conversely, the CD11b activator LA1 reduced chondrocyte mineralization, Alp expression, IL-6 production and collagen X expression. In the meniscectomy (MNX) model of murine knee osteoarthritis, deficiency of CD11b led to more severe OA (OARSI scoring of medial cartilage damage in CD11b: 5.6 ± 1.8, in WT: 1.2 ± 0.5, p < 0.05, inflammation in CD11b: 2.8 ± 0.2, in WT: 1.4 ± 0.5). In conclusion, these data demonstrate that CD11b signaling prevents chondrocyte hypertrophy and chondrocyte mineralization in vitro and has a protective role in models of OA in vivo.

The role of the gasotransmitter hydrogen sulfide in pathological calcification.

Calcification is the deposition of minerals, mainly hydroxyapatite, inside the cell or in the extracellular matrix. Physiological calcification is central for many aspects of development including skeletal and tooth growth; conversely, pathological mineralization occurs in soft tissues and is significantly associated with malfunction and impairment of the tissue where it is located. Various mechanisms have been proposed to explain calcification. However, this research area lacks a more integrative, systemic, and global perspective that could explain both physiological and pathological processes. In this review, we propose such an integrated explanation. Hydrogen sulfide (H2 S) is a newly recognized multifunctional gasotransmitters and tis actions have been studied in different physiological and pathological contexts, but little is known about its potential role on calcification. Interestingly, we found that H2 S promotes calcification under physiological conditions and has an inhibitory effect on pathological processes. This makes H2 S a potential therapy for diseases related to pathological calcification. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Targeted knock-in mice expressing the oxidase-fixed form of xanthine oxidoreductase favor tumor growth.

Xanthine oxidoreductase has been implicated in cancer. Nonetheless, the role played by its two convertible forms, xanthine dehydrogenase (XDH) and oxidase (XO) during tumorigenesis is not understood. Here we produce XDH-stable and XO-locked knock-in (ki) mice to address this question. After tumor transfer, XO ki mice show strongly increased tumor growth compared to wild type (WT) and XDH ki mice. Hematopoietic XO expression is responsible for this effect. After macrophage depletion, tumor growth is reduced. Adoptive transfer of XO-ki macrophages in WT mice increases tumor growth. In vitro, XO ki macrophages produce higher levels of reactive oxygen species (ROS) responsible for the increased Tregs observed in the tumors. Blocking ROS in vivo slows down tumor growth. Collectively, these results indicate that the balance of XO/XDH plays an important role in immune surveillance of tumor development. Strategies that inhibit the XO form specifically may be valuable in controlling cancer growth.

TRANSFORMING GROWTH FACTOR BETA HAS DUAL EFFECTS ON MMP9 AND uPA EXPRESSION IN HTR-8/SVneo HUMAN TROPHOBLASTIC CELL LINE / El factor de crecimiento transformante beta tiene efecto dual en la expresión de MMP9 y uPA en la línea celular de trofoblasto _HTR-8/SVneo

ABSTRACT Invasion of trophoblast into endometrium is vital for successful pregnancy development. MMP9 and uPA are key proteases in this process, but it is still not clear the regulation of its expression by Transforming Growth Factor Beta (TGF-β), a known negative regulator of trophoblast invasion. We evaluated the effect of TGF-β on the transcriptional expression of uPA and MMP9 over time, in HTR-8/SVneo trophoblast cells cultured with or without 0.5 % fetal bovine serum, via RT qPCR. The involved transcription factors and signaling pathways were analyzed in silico, using Proscan, Enrich, PCViz and Wiki Pathway. Results showed that TGF-β temporarily regulates the expression of uPA and MMP9. Serum modified the nature of TGF-β's effects on uPA expression, from negative without serum to positive with it, showing opposite effects on MMP9 expression. In silico analysis evidenced different transcription factors for each protease, some belonging to TGF-β signaling pathway, and crosstalk with MAPK and Wnt/β -catenin pathways. The TGF-β dual role is discussed proposing that serum affects the cellular context. Transcriptional regulation of MMP9 and uPA by TGF-β is differential and depends on serum presence and evaluation time.

RESUMEN La invasión del trofoblasto al endometrio es vital para el correcto desarrollo del embarazo. Las proteasas MMP9 y uPA son claves en este proceso, pero aún no es clara la regulación de su expresión por parte del Factor de Crecimiento Transformante beta (TGF-β), conocido por sus acciones no invasivas sobre el trofoblasto. En este trabajo evaluamos el efecto del TGF-β sobre la expresión transcripcional de uPA y MMP9 en células de la línea de trofoblasto HTR-8/SVneo cultivadas con o sin suero fetal bovino al 0,5 %, mediante RT qPCR. Se analizaron in sillico los potenciales factores de transcripción y vías de señalización involucradas empleando Proscan, Enrich, PCViz y WikiPathway. Los resultados muestran que el TGF-β regula temporalmente la expresión de uPA y MMP9. El suero modificó la naturaleza del efecto del TGF-β sobre la expresión de uPA, de negativo en ausencia de suero a positivo en presencia de este, presentando efectos opuestos para la expresión de MMP9. El análisis in sillico evidenció diferentes factores de transcripción para cada proteasa, algunos pertenecientes a la vía de señalización del TGF-β, y un entrecruzamiento con la vía MAPK y Wnt/β-catenina. Los resultados sugieren que la regulación transcripcional de MMP9 y uPA por parte del TGF-β es diferencial y depende de la presencia de suero y tiempo de evaluación.

Hydrogen sulfide inhibits NLRP3 inflammasome activation and reduces cytokine production both in vitro and in a mouse model of inflammation.

A variety of stimuli, including monosodium urate (MSU) crystals, activate the NLRP3 inflammasome, and this activation involves several molecular mechanisms including xanthine oxidase (XO) up-regulation and mitochondrial dysfunction. Upon oligomerization of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 becomes active and cleaves the proinflammatory cytokine IL-1ß into its active secreted form. Hydrogen sulfide (H2S), a gasotransmitter mainly produced by cystathionine γ-lyase (CSE) in macrophages, could modulate inflammation. Here, we sought to investigate the effects of exogenous and endogenous H2S on NLRP3 inflammasome activation in vitro and in vivo Primed bone marrow-derived macrophages (BMDM) isolated from wildtype (wt) or CSE-deficient mice and human macrophages (THP1 cells and primary macrophages), were stimulated with MSU crystals in the presence or absence of a H2S donor, sodium thiosulfate (STS) or GYY4137 (GYY). In murine and human macrophages in vitro, both STS and GYY inhibited MSU crystal-induced IL-1ß secretion in a dose-dependent manner. Moreover, the H2S donors inhibited MSU crystal-induced XO/caspase-1 activities, mitochondrial reactive oxygen species (ROS) generation, and ASC oligomerization. Accordingly, IL-1ß secretion and XO/caspase-1 activities were higher in CSE-deficient BMDMs than in wt BMDMs. For in vivo studies, we experimentally induced peritonitis by intraperitoneal injection of MSU crystals into mice. GYY pretreatment ameliorated inflammation, evidenced by decreased IL-6/monocyte chemoattractant protein-1 (MCP-1) released into peritoneal lavages. Taken together, our results suggest that both exogenous (via H2S donors) and endogenous (via CSE) H2S production may represent approaches for managing, for example, acute gout or other inflammation conditions.