Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts

Introduction

Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts.

Methods and Results

We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells) were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts.

Conclusions

We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival fibroblasts, and suggest that gingival fibroblasts may have an ECM-degrading phenotype during conditions of hyperleptinaemia (e.g., obesity, type 2 diabetes mellitus, exogenous leptin therapy).     

The extraction and assay of aminoacyl-transfer-ribonucleic acid synthetases of tobacco leaf

1. Aminoacyl-transfer-RNA synthetase activity in extracts prepared from tobacco leaf was increased 3–5-fold when sodium thioglycollate (30mm) and magnesium chloride (16mm) were included in the extraction medium. Omitting sucrose (0·45m) from the extraction medium did not alter the activity. 2. Activity was a linear function of enzyme concentration up to 1 disk (30mg. fresh wt.)/ml. and was not affected by dialysis at any concentration. 3. Activity increased about 13-fold above control values when a mixture of 21 amino acids and amides (1mm) was added to the reaction mixture. 4. Under the conditions used in the standard assay for aminoacyl-transfer-RNA synthetase activity K_m (ATP) was 0·65mm and K_m (l-amino acids) was 70μm. 5. Activity above the control value was found with all amino acids and amides tested except alanine, arginine, glutamic acid, glutamine and hydroxyproline. Activity was highest with leucine, isoleucine, valine, cysteine and histidine. Total activity with a mixture of 21 amino acids and amides was 20% lower than the total activity of the enzymes assayed separately.     

Morphological and physiological characteristics of a non-heterocystous strain of the cyanobacterium Mastigocladus laminosus Cohn from fumarolic soil on Mt Erebus,Antarctica

Summary An unusual strain of the thermophilic cyanobacterium Mastigocladus laminosus occurs on warm soils on the volcano Mt Erebus (77°32S, 167°8E). It differs morphologically from the two genetically distinct forms described from thermal habitats elsewhere. Heterocysts are lacking and true-branching is rare. Its photosynthetic rate, and the contrasting rates of two less thermotolerant algae from Mt Erebus soils, Phormidium fragile (Cyanobacteria) and Pseudococcomyxa simplex (Chlorophyta), were measured over the range -2° to 62°C. The optimum temperature range of M. laminosus was 35° to 50°C. Photosynthetic response to temperature of all three algae in the laboratory correlated well with distribution patterns in the field, confirming that zonation patterns were temperature controlled. M. laminosus retained viability following exposure to deep-freezing, freeze-thaw cycles and desiccation. Viability of the alga in culture was lost following exposure to 50°C in darkness for 42 days and following 42 days in the light at 0°C. Discussion suggests the alga would survive long distance airborne dispersal in the desiccated condition but would not survive the duration of overwinter darkness on moist soils at the warmer end of its range of occurrence in the field.     

Presynaptic alpha-receptor control of adrenergic transmitter release in blood vessels

The presynaptic alpha-adrenergic receptor control of transmitter release in vascular tissues is discussed. A model of adrenergic innervation of the vascular bed is proposed based on ultrastructural and histochemical evidence. Evidence is presented to support the concept of intermittent or periodic release of norepinephrine (NE) from the varicosity. Intermittency combined with a mechanism such as presynaptic control to ensure spatial distribution of release sites, along with a slow effector response and recovery, results in a smooth, generalized change in tone and an overall economy of transmitter. The effective concentration of NE around the presynaptic membrane is maintained for considerably less than 0.1 s. It is argued that the transient presence of transmitter in the synapse combined with intermittency of release does not favor accumulation of transmitter at the cleft at physiological frequencies or desensitization of presynaptic receptors. In addition, intermittency provides an explanation for why exogenous NE is more effective presynaptically in influencing release than endogenous NE. The importance of cleft width in presynaptic control of transmitter release, the possible complications caused by facilitation, and resolution of some apparent problems with the presynaptic hypothesis are also discussed.     

The histochemical specificity of High Iron Diamine—Alcian Blue

Summary The specificity of the High Iron Diamine—Alcian Blue pH2.5 (HID—AB 2.5) procedure was examined in tissue sites containing sialogycoproteins alone or differing proportions of sialo- and sulphosialoglycoproteins. Studies with HID in differing final concentrations of hydrochloric acid or sodium chloride confirmed that staining is dependent upon both the pH and the ionic strength of the dye bath and demonstrated a marked heterogeneity in the pKa of the anionic groups of sialosulphoglycoproteins. Use of the sequence High Iron Diamine—Alcian Blue pH 1.0 demonstrated that complete or almost complete staining ofO-sulphate esters occurred when HID was prepared in water (final pH 1.3). However, under these conditions HID—AB 2.5 was shown to be non-specific because only black HID staining was observed in sites containing large quantities of sialic acids. This non-specificity was due either to the masking of Alcian Blue staining by HID and/or the black HID staining of anionic groups other than sulphate. These results may account for some of the conflicting data obtained by different groups of investigators who have studied transitional mucosa in human colonic diseases. Caution should be used in drawing conclusions from the use of HID—AB 2.5 without confirmatory evidence from other more specific procedures.     

Disequilibrium,Adaptation, and the Norse Settlement of Greenland

There is increasing evidence to suggest that arctic cultures and ecosystems have followed non-linear responses to climate change. Norse Scandinavian farmers introduced agriculture to sub-arctic Greenland in the late tenth century, creating synanthropic landscapes and utilising seasonally abundant marine and terrestrial resources. Using a niche-construction framework and data from recent survey work, studies of diet, and regional-scale climate proxies we examine the potential mismatch between this imported agricultural niche and the constraints of the environment from the tenth to the fifteenth centuries. We argue that landscape modification conformed the Norse to a Scandinavian style of agriculture throughout settlement, structuring and limiting the efficacy of seasonal hunting strategies. Recent climate data provide evidence of sustained cooling from the mid thirteenth century and climate variation from the early fifteenth century. Archaeological evidence suggests that the Norse made incremental adjustments to the changing sub-arctic environment, but were limited by cultural adaptations made in past environments.     

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion

Objective and backgroundActivation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice.MethodsPlasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury.ResultsIn patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects.ConclusionHMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.     

Mechanistic overview of reactive species-induced degradation of the endothelial glycocalyx during hepatic ischemia/reperfusion injury

Endothelial cells are covered by a delicate meshwork of glycoproteins known as the glycocalyx. Under normophysiological conditions the glycocalyx plays an active role in maintaining vascular homeostasis by deterring primary and secondary hemostasis and leukocyte adhesion and by regulating vascular permeability and tone. During (micro)vascular oxidative and nitrosative stress, which prevails in numerous metabolic (diabetes), vascular (atherosclerosis, hypertension), and surgical (ischemia/reperfusion injury, trauma) disease states, the glycocalyx is oxidatively and nitrosatively modified and degraded, which culminates in an exacerbation of the underlying pathology. Consequently, glycocalyx degradation due to oxidative/nitrosative stress has far-reaching clinical implications. In this review the molecular mechanisms of reactive oxygen and nitrogen species-induced destruction of the endothelial glycocalyx are addressed in the context of hepatic ischemia/reperfusion injury as a model disease state. Specifically, the review focuses on (i) the mechanisms of glycocalyx degradation during hepatic ischemia/reperfusion, (ii) the molecular and cellular players involved in the degradation process, and (iii) its implications for hepatic pathophysiology. These topics are projected against a background of liver anatomy, glycocalyx function and structure, and the biology/biochemistry and the sources/targets of reactive oxygen and nitrogen species. The majority of the glycocalyx-related mechanisms elucidated for hepatic ischemia/reperfusion are extrapolatable to the other aforementioned disease states.