Co-Culture of P. gingivalis and F. nucleatum Synergistically Elevates IL-6 Expression via TLR4 Signaling in Oral Keratinocytes.

Periodontitis, characterized by persistent inflammation in the periodontium, is intricately connected to systemic diseases, including oral cancer. Bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, play a pivotal role in periodontitis development because they contribute to dysbiosis and tissue destruction. Thus, comprehending the interplay between these bacteria and their impacts on inflammation holds significant relevance in clinical understanding and treatment advancement. In the present work, we explored, for the first time, their impacts on the expressions of pro-inflammatory mediators after infecting oral keratinocytes (OKs) with a co-culture of pre-incubated P. gingivalis and F. nucleatum. Our results show that the co-culture increases IL-1ß, IL-8, and TNF-α expressions, synergistically augments IL-6, and translocates NF-kB to the cell nucleus. These changes in pro-inflammatory mediators-associated with chronic inflammation and cancer-correlate with an increase in cell migration following infection with the co-cultured bacteria or P. gingivalis alone. This effect depends on TLR4 because TLR4 knockdown notably impacts IL-6 expression and cell migration. Our study unveils, for the first time, crucial insights into the outcomes of their co-culture on virulence, unraveling the role of bacterial interactions in polymicrobial diseases and potential links to oral cancer.

Dietary Potassium Downregulates Angiotensin-I Converting Enzyme, Renin, and Angiotensin Converting Enzyme 2.

BACKGROUND: The importance of dietary potassium in health and disease has been underestimated compared with that placed on dietary sodium. Larger effort has been made on reduction of sodium intake and less on the adequate dietary potassium intake, although natural food contains much more potassium than sodium. The benefits of a potassium-rich diet are known, however, the mechanism by which it exerts its preventive action, remains to be elucidated. With the hypothesis that dietary potassium reduces renal vasoconstrictor components of the renin-angiotensin system in the long-term, we studied the effect of high potassium diet on angiotensin-I converting enzyme, renin, and angiotensin converting enzyme 2. METHODS: Sprague Dawley male rats on a normal sodium diet received normal potassium (0.9%, NK) or high potassium diet (3%, HK) for 4 weeks. Urine was collected in metabolic cages for electrolytes and urinary volume measurement. Renal tissue was used to analyze angiotensin-I converting enzyme, renin, and angiotensin converting enzyme 2 expression. Protein abundance analysis was done by Western blot; gene expression by mRNA levels by RT-qPCR. Renal distribution of angiotensin-I converting enzyme and renin was done by immunohistochemistry and morphometric analysis in coded samples. RESULTS: High potassium diet (4 weeks) reduced the levels of renin, angiotensin-I converting enzyme, and angiotensin converting enzyme 2. Angiotensin-I converting enzyme was located in the brush border of proximal tubules and with HK diet decreased the immunostaining intensity (P < 0.05), decreased the mRNA (P < 0.01) and the protein levels (P < 0.01). Renin localization was restricted to granular cells of the afferent arteriole and HK diet decreased the number of renin positive cells (P < 0.01) and renin mRNA levels (P < 0.01). High potassium intake decreased angiotensin converting enzyme 2 gene expression and protein levels (P < 0.01).No morphological abnormalities were observed in renal tissue during high potassium diet.The reduced expression of angiotensin-I converting enzyme, renin, and angiotensin converting enzyme 2 during potassium supplementation suggest that high dietary potassium intake could modulate these vasoactive enzymes and this effects can contribute to the preventive and antihypertensive effect of potassium.

Imbalance in Renal Vasoactive Enzymes Induced by Mild Hypoxia: Angiotensin-Converting Enzyme Increases While Neutral Endopeptidase Decreases.

Chronic hypoxia has been postulated as one of the mechanisms involved in salt-sensitive hypertension and chronic kidney disease (CKD). Kidneys have a critical role in the regulation of arterial blood pressure through vasoactive systems, such as the renin-angiotensin and the kallikrein-kinin systems, with the angiotensin-converting enzyme (ACE) and kallikrein being two of the main enzymes that produce angiotensin II and bradykinin, respectively. Neutral endopeptidase 24.11 or neprilysin is another enzyme that among its functions degrade vasoactive peptides including angiotensin II and bradykinin, and generate angiotensin 1-7. On the other hand, the kidneys are vulnerable to hypoxic injury due to the active electrolyte transportation that requires a high oxygen consumption; however, the oxygen supply is limited in the medullary regions for anatomical reasons. With the hypothesis that the chronic reduction of oxygen under normobaric conditions would impact renal vasoactive enzyme components and, therefore; alter the normal balance of the vasoactive systems, we exposed male Sprague-Dawley rats to normobaric hypoxia (10% O2) for 2 weeks. We then processed renal tissue to identify the expression and distribution of kallikrein, ACE and neutral endopeptidase 24.11 as well as markers of kidney damage. We found that chronic hypoxia produced focal damage in the kidney, mainly in the cortico-medullary region, and increased the expression of osteopontin. Moreover, we observed an increase of ACE protein in the brush border of proximal tubules at the outer medullary region, with increased mRNA levels. Kallikrein abundance did not change significantly with hypoxia, but a tendency toward reduction was observed at protein and mRNA levels. Neutral endopeptidase 24.11 was localized in proximal tubules, and was abundantly expressed under normoxic conditions, which markedly decreased both at protein and mRNA levels with chronic hypoxia. Taken together, our results suggest that chronic hypoxia produces focal kidney damage along with an imbalance of key components of the renal vasoactive system, which could be the initial steps for a long-term contribution to salt-sensitive hypertension and CKD.

Regulation of cardiac autophagy by insulin-like growth factor 1.

Insulin-like growth factor-1 (IGF-1) signaling is a key pathway in the control of cell growth and survival. Three critical nodes in the IGF-1 signaling pathway have been described in cardiomyocytes: protein kinase Akt/mammalian target of rapamycin (mTOR), Ras/Raf/extracellular signal-regulated kinase (ERK), and phospholipase C (PLC)/inositol 1,4,5-triphosphate (InsP3 )/Ca(2+) . The Akt/mTOR and Ras/Raf/ERK signaling arms govern survival in the settings of cardiac stress and hypertrophic growth. By contrast, PLC/InsP3 /Ca(2+) functions to regulate metabolic adaptability and gene transcription. Autophagy is a catabolic process involved in protein degradation, organelle turnover, and nonselective breakdown of cytoplasmic components during nutrient starvation or stress. In the heart, autophagy is observed in a variety of human pathologies, where it can be either adaptive or maladaptive, depending on the context. We proposed the hypothesis that IGF-1 protects the heart by rescuing the mitochondrial metabolism and the energetics state, reducing cell death and controls the potentially exacerbate autophagic response to nutritional stress. In light of the importance of IGF-1 and autophagy in the heart, we review here IGF-1 signaling and autophagy regulation in the context of cardiomyocyte nutritional stress.

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains.

Glucose deprivation causes oxidative stress and stimulates aggresome formation and autophagy in cultured cardiac myocytes.

Aggresomes are dynamic structures formed when the ubiquitin-proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6 h. However, at longer times (18 h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with gamma-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.

Iron induces protection and necrosis in cultured cardiomyocytes: Role of reactive oxygen species and nitric oxide.

We investigate here the role of reactive oxygen species and nitric oxide in iron-induced cardiomyocyte hypertrophy or cell death. Cultured rat cardiomyocytes incubated with 20 microM iron (added as FeCl(3)-Na nitrilotriacetate, Fe-NTA) displayed hypertrophy features that included increased protein synthesis and cell size, plus realignment of F-actin filaments along with sarcomeres and activation of the atrial natriuretic factor gene promoter. Incubation with higher Fe-NTA concentrations (100 microM) produced cardiomyocyte death by necrosis. Incubation for 24 h with Fe-NTA (20-40 microM) or the nitric oxide donor Delta-nonoate increased iNOS mRNA but decreased iNOS protein levels; under these conditions, iron stimulated the activity and the dimerization of iNOS. Fe-NTA (20 microM) promoted short- and long-term generation of reactive oxygen species, whereas preincubation with l-arginine suppressed this response. Preincubation with 20 microM Fe-NTA also attenuated the necrotic cell death triggered by 100 microM Fe-NTA, suggesting that these preincubation conditions have cardioprotective effects. Inhibition of iNOS activity with 1400 W enhanced iron-induced ROS generation and prevented both iron-dependent cardiomyocyte hypertrophy and cardioprotection. In conclusion, we propose that Fe-NTA (20 microM) stimulates iNOS activity and that the enhanced NO production, by promoting hypertrophy and enhancing survival mechanisms through ROS reduction, is beneficial to cardiomyocytes. At higher concentrations, however, iron triggers cardiomyocyte death by necrosis.

Regulatory volume decrease in cardiomyocytes is modulated by calcium influx and reactive oxygen species.

We investigated the role of Ca(2+) in generating reactive oxygen species (ROS) induced by hyposmotic stress (Hypo) and its relationship to regulatory volume decrease (RVD) in cardiomyocytes. Hypo-induced increases in cytoplasmic and mitochondrial Ca(2+). Nifedipine (Nife) inhibited both Hypo-induced Ca(2+) and ROS increases. Overexpression of catalase (CAT) induced RVD and a decrease in Hypo-induced blebs. Nife prevented CAT-dependent RVD activation. These results show a dual role of Hypo-induced Ca(2+) influx in the control of cardiomyocyte viability. Hypo-induced an intracellular Ca(2+) increase which activated RVD and inhibited necrotic blebbing thus favoring cell survival, while simultaneously increasing ROS generation, which in turn inhibited RVD and induced necrosis.

Ceramide-induced formation of ROS and ATP depletion trigger necrosis in lymphoid cells.

In lymphocytes, Fas activation leads to both apoptosis and necrosis, whereby the latter form of cell death is linked to delayed production of endogenous ceramide and is mimicked by exogenous administration of long- and short-chain ceramides. Here molecular events associated with noncanonical necrotic cell death downstream of ceramide were investigated in A20 B lymphoma and Jurkat T cells. Cell-permeable, C6-ceramide (C6), but not dihydro-C6-ceramide (DH-C6), induced necrosis in a time- and dose-dependent fashion. Rapid formation of reactive oxygen species (ROS) within 30 min of C6 addition detected by a dihydrorhodamine fluorescence assay, as well as by electron spin resonance, was accompanied by loss of mitochondrial membrane potential. The presence of N-acetylcysteine or ROS scavengers like Tiron, but not Trolox, attenuated ceramide-induced necrosis. Alternatively, adenovirus-mediated expression of catalase in A20 cells also attenuated cell necrosis but not apoptosis. Necrotic cell death observed following C6 exposure was associated with a pronounced decrease in ATP levels and Tiron significantly delayed ATP depletion in both A20 and Jurkat cells. Thus, apoptotic and necrotic death induced by ceramide in lymphocytes occurs via distinct mechanisms. Furthermore, ceramide-induced necrotic cell death is linked here to loss of mitochondrial membrane potential, production of ROS, and intracellular ATP depletion.

Reactive oxygen species inhibit hyposmotic stress-dependent volume regulation in cultured rat cardiomyocytes.

Cells have developed compensatory mechanisms to restore cell volume, and the ability to resist osmotic swelling or shrinkage parallels their resistance to necrosis or apoptosis. There are several mechanisms by which cells adapt to hyposmotic stress including that of regulatory volume decrease. In ischemia and reperfusion, cardiomyocytes are exposed to hyposmotic stress, but little is known as to how their volume is controlled. Exposure of cultured neonatal rat cardiomyocytes to hyposmotic media induced a rapid swelling without any compensatory regulatory volume decrease. The hyposmotic stress increased the production of reactive oxygen species, mainly through NADPH oxidase. Adenoviral overexpression of catalase inhibited the hyposmosis-dependent OH(*) production, induced the regulatory volume decrease mechanism, and prevented cell death. These results suggest that hyposmotic stress of cardiomyocytes stimulates production of reactive oxygen species which are closely linked to volume regulation and cell death.