Magnesium Inhibits Wnt/β-Catenin Activity and Reverses the Osteogenic Transformation of Vascular Smooth Muscle Cells

Magnesium reduces vascular smooth muscle cell (VSMC) calcification in vitro but the mechanism has not been revealed so far. This work used only slightly increased magnesium levels and aimed at determining: a) whether inhibition of magnesium transport into the cell influences VSMC calcification, b) whether Wnt/β-catenin signaling, a key mediator of osteogenic differentiation, is modified by magnesium and c) whether magnesium can influence already established vascular calcification. Human VSMC incubated with high phosphate (3.3 mM) and moderately elevated magnesium (1.4 mM) significantly reduced VSMC calcification and expression of the osteogenic transcription factors Cbfa-1 and osterix, and up-regulated expression of the natural calcification inhibitors matrix Gla protein (MGP) and osteoprotegerin (OPG). The protective effects of magnesium on calcification and expression of osteogenic markers were no longer observed in VSMC cultured with an inhibitor of cellular magnesium transport (2-aminoethoxy-diphenylborate [2-APB]). High phosphate induced activation of Wnt/β-catenin pathway as demonstrated by the translocation of β-catenin into the nucleus, increased expression of the frizzled-3 gene, and downregulation of Dkk-1 gene, a specific antagonist of the Wnt/β-catenin signaling pathway. The addition of magnesium however inhibited phosphate-induced activation of Wnt/β-catenin signaling pathway. Furthermore, TRPM7 silencing using siRNA resulted in activation of Wnt/β-catenin signaling pathway. Additional experiments were performed to test the ability of magnesium to halt the progression of already established VSMC calcification in vitro. The delayed addition of magnesium decreased calcium content, down-regulated Cbfa-1 and osterix and up-regulated MGP and OPG, when compared with a control group. This effect was not observed when 2-APB was added. In conclusion, magnesium transport through the cell membrane is important to inhibit VSMC calcification in vitro. Inhibition of Wnt/β-catenin by magnesium is one potential intracellular mechanism by which this anti-calcifying effect is achieved

Pflanzlicher Lichtsammler (LHCII) in Hybridkomplexen mit organischen Farbstoffen und anorganischen Halbleiter-Nanokristallen (Quantum dots)

Der Lichtsammelkomplex II (LHCII) höherer Pflanzen ist eines der häufigsten Membranproteine der Welt. Er bindet 14 Chlorophylle und 4 Carotinoide nicht kovalent und fungiert in vivo als Lichtantenne des Photosystems II. Eine optimale Absorption von Licht ist auch bei Solarzellen entscheidend und es liegt nahe hier dasselbe Prinzip zu verwenden. Dafür bietet sich der Einsatz biologischer Komponenten wie des LHCII an. Dieser wurde evolutionär für eine effektive Absorption und Weiterleitung von Sonnenenergie optimiert. Zusätzlich lässt er sich in vitro in rekombinanter Form rekonstituieren. Für eine eventuelle Nutzung des LHCII in technologischen Anwendungen bedarf es der Interaktion mit anderen, vorzugsweise synthetischen Komponenten. Daher wurde die Bindung und der Energietransfer zwischen dem LHCII und organischen Fluoreszenzfarbstoffen sowie anorganischen „Quantum dots“ (QDs) untersucht. rnMit Donorfarbstoffen wurde die Grünlücke des LHCII funktionell geschlossen. Dafür wurden bis zu vier Fluoreszenzfarbstoffe kovalent an den LHCII gebunden. Diese Interaktion erfolgte sowohl mit Maleimiden an Cysteinen als auch mit N-Hydroxysuccinimidylestern an Lysinen. Die Assemblierung, Struktur und Funktion des Pigment-Protein-Komplexes wurde durch die Fluoreszenzfarbstoffe nicht gestört.rnAuf der Suche nach einem Farbstoff, der als Akzeptor die vom LHCII aufgenommene Energie übernimmt und durch Elektronenabgabe in elektrische Energie umwandelt, wurden drei Rylenfarbstoffe, ein Quaterrylen und zwei Terrylene, untersucht. Der LHCII konnte mit allen Farbstoffen erfolgreich markiert werden. Für die Nutzung der Hybridkomplexe ergaben sich allerdings Probleme. Das Quaterrylen beeinträchtigte aufgrund seiner Hydrophobizität die Rekonstitution des Proteins, während bei beiden Terrylenen der Energietransfer ineffizient war.rn Zusätzlich zu den Standard-Verknüpfungen zwischen Farbstoffen und Proteinen wurde in dieser Arbeit die „native chemische Ligation“ etabliert. Hierfür wurde eine LHCII-Mutante mit N-terminalem Cystein hergestellt, markiert und rekonstituiert. Messdaten an diesem Hybridkomplex ließen auf einen Energietransfer zwischen Farbstoff und Protein schließen. rnIn Hybridkomplexen sollen langfristig zur Ladungstrennung fähige Typ II-QDs Anwendung finden, wobei der LHCII als Lichtantenne dienen soll. Bis diese QDs verwendet werden können, wurden grundlegende Fragen der Interaktion beider Materialen an Typ I-QDs mit Energietransfer zum LHCII untersucht. Dabei zeigte sich, dass QDs in wässriger Lösung schnell aggregieren und entsprechende Kontrollen wichtig sind. Weiterführend konnte anhand der Trennung von ungebundenem und QD-gebundenem LHCII die Bindung von LHCII an QDs bestätigt werden. Dabei wurden Unterschiede in der Bindungseffizienz in Abhängigkeit der verwendeten LHCII und QDs festgestellt. Durch Herstellung von Fusionsproteinen aus LHCII und Affinitätspeptiden konnte die Bindung optimiert werden. Ein Energietransfer von QDs zu LHCII war nicht sicher nachzuweisen, da in den Hybridkomplexen zwar die QD- (Donor-) Fluoreszenz gelöscht, aber die LHCII- (Akzeptor-) Fluoreszenz nicht entsprechend stimuliert wurde.rnZusammenfassend wurden in dieser Arbeit einige Hybridkomplexe hergestellt, die in weiterführenden Ansätzen Verwendung finden können. Auf die hier gewonnenen Erkenntnisse über Interaktionen zwischen LHCII und synthetischen Materialien kann jetzt weiter aufgebaut werden.The light-harvesting complex II (LHCII) is one of the most abundant membrane proteins. It binds non-covalently 14 chlorophylls and 4 carotenoids and acts in vivo as a light-antenna for Photosystem II. Optimal light absorption is also desirable in solar cells. This can possibly be achieved by using biological components like LHCII. This complex is not only evolutionary optimised for this purpose but furthermore it can be reconstituted in vitro. In such applications, LHCII would have to interact with synthetic components. Therefore, the binding and energy transfer between LHCII, organic dyes and inorganic quantum dots (QDs) were analysed in this work.rnThe absorption “green gap” of the LHCII was functionally filled in by covalently attaching up to four donor dyes. Dye maleimides were bound to cystein residues and N-hydroxysuccinimide esters to lysin residues. Neither modification interfered with the assembly, structure and function of the pigment-protein-complex.rnThree rylene dyes, a quaterrylene and two terrylenes, were examined for their suitability to act as acceptors of the LHCII-absorbed energy. All dyes were successfully bound to the protein, however the hybrid complexes did not turn out to be useful. The hydrophobicity of the quaterrylene disturbed protein folding, whereas the energy transfer was inefficient with both terrylenes.rnAs an additional labelling technique, the “native chemical ligation” was established for the LHCII. A new mutant with an N-terminal cysteine was produced, labelled and reconstituted and the data suggested an energy transfer between dye and protein.rnType-II QDs are capable of charge transfer and they shall be integrated with the LHCII in hybrid complexes. Until they can be used, basic questions regarding the interaction of both materials were analysed by using type-I QDs acting as energy donors for the LHCII. It was shown that QDs tend to aggregate in aqueous solution and corresponding controls are necessary. The binding of both components was verified by separating unlabelled from QD-bound LHCII. Furthermore, differences in binding efficiencies depending on the components used were detected by this technique. Newly synthesised fusion proteins of LHCII and affinity peptides optimised the binding. The QD (donor) fluorescence was quenched in hybrid complexes; however a corresponding sensitised LHCII (acceptor) fluorescence was not detected. Therefore an energy transfer could not be verified so far.rnIn summary, several usable hybrid complexes were produced and the new information about interactions between LHCII and synthetic materials will lead to the construction of applicable hybrids

Site-specific incorporation of perylene into an N-terminally modified light-harvesting complex II

Employing the utility of the native chemical ligation, site-specific attachment of an ultrastable perylene dye to a derivative of the major light-harvesting complex (LHCII) was demonstrated. Biochemical analysis of the conjugate indicated that the structure and function of LHCII remain largely unaffected by the N-terminal modification.

Altered serum levels of FGF-23 and magnesium are independent risk factors for an increased albumin-to-creatinine ratio in type 2 diabetics with chronic kidney disease

Aims: To investigate the role of FGF-23 and magnesium in relation to the albumin-to-creatinine ratio in type 2 diabetics with chronic kidney disease (CKD) stages 2-4.Methods: In a cross-sectional study we included all eligible type 2 diabetic patients with CKD stages 2-4, followed in our outpatient Diabetic Kidney clinic. We used descriptive statistics, the Student's t-test, ANOVA and the chi-square tests. Our population was divided according to the UACR (G1 30-300 mg/g and G2 >= 300 mg/g), and compared these groups regarding several biological and laboratorial parameters. We employed a multiple regression model to identify risk factors of increased UACR.Results: The patients in G2 displayed a lower eGFR (p = 0.0001) and, had lower levels of magnesium (p = 0.004) as well as higher levels of FGF-23 (p = 0.043) compared to patients in Gl.FGF-23 (beta = 0.562, P = 0.0001) and the magnesium (beta = - 8.916, p = 0.0001) were associated with increased UACR.Conclusions: A dysregulation of mineral metabolism, reflected by altered levels of magnesium and FGF-23, correlates with an increased UACR in type 2 diabetic patients with CKD stages 2-4. (C) 2016 Elsevier Inc. All rights reserved

Low Magnesium Levels and FGF-23 Dysregulation Predict Mitral Valve Calcification as well as Intima Media Thickness in Predialysis Diabetic Patients

Background. Mitral valve calcification and intima media thickness (IMT) are common complications of chronic kidney disease (CKD) implicated with high cardiovascular mortality. Objective. To investigate the implication of magnesium and fibroblast growth factor-23 (FGF-23) levels with mitral valve calcification and IMT in CKD diabetic patients. Methods. Observational, prospective study involving 150 diabetic patients with mild to moderate CKD, divided according to Wilkins Score. Carotid-echodoppler and transthoracic echocardiography were used to assess calcification. Statistical tests used to establish comparisons between groups, to identify risk factors, and to establish cut-off points for prediction of mitral valve calcification. Results. FGF-23 values continually increased with higher values for both IMT and calcification whereas the opposite trend was observed for magnesium. FGF-23 and magnesium were found to independently predict mitral valve calcification and IMT (P<0.05). Using Kaplan-Meier analysis, the number of deaths was higher in patients with lower magnesium levels and poorer Wilkins score. The mean cut-off value for FGF-23 was 117 RU/mL and for magnesium 1.7 mg/dL. Conclusions. Hypomagnesemia and high FGF-23 levels are independent predictors of mitral valve calcification and IMT and are risk factors for cardiovascular mortality in this population. They might be used as diagnostic/therapeutic targets in order to better manage the high cardiovascular risk in CKD patients

Effect of a magnesium-based phosphate binder on medial calcification in a rat model of uremia

Calcium-based phosphate binders are used to control hyperphosphatemia; however, they promote hypercalcemia and may accelerate aortic calcification. Here we compared the effect of a phosphate binder containing calcium acetate and magnesium carbonate (CaMg) to that of sevelamer carbonate on the development of medial calcification in rats with chronic renal failure induced by an adenine diet for 4 weeks. After 1 week, rats with chronic renal failure were treated with vehicle, 375 or 750mg/kg CaMg, or 750mg/kg sevelamer by daily gavage for 5 weeks. Renal function was significantly impaired in all groups. Vehicle-treated rats with chronic renal failure developed severe hyperphosphatemia, but this was controlled in treated groups, particularly by CaMg. Neither CaMg nor sevelamer increased serum calcium ion levels. Induction of chronic renal failure significantly increased serum PTH, dose-dependently prevented by CaMg but not sevelamer. The aortic calcium content was significantly reduced by CaMg but not by sevelamer. The percent calcified area of the aorta was significantly lower than vehicle-treated animals for all three groups. The presence of aortic calcification was associated with increased sox9, bmp-2, and matrix gla protein expression, but this did not differ in the treatment groups. Calcium content in the carotid artery was lower with sevelamer than with CaMg but that in the femoral artery did not differ between groups. Thus, treatment with either CaMg or sevelamer effectively controlled serum phosphate levels in CRF rats and reduced aortic calcification

A magnesium based phosphate binder reduces vascular calcification without affecting bone in chronic renal failure rats

The alternative phosphate binder calcium acetate/magnesium carbonate (CaMg) effectively reduces hyperphosphatemia, the most important inducer of vascular calcification, in chronic renal failure (CRF). In this study, the effect of low dose CaMg on vascular calcification and possible effects of CaMg on bone turnover, a persistent clinical controversy, were evaluated in chronic renal failure rats. Adenine-induced CRF rats were treated daily with 185 mg/kg CaMg or vehicle for 5 weeks. The aortic calcium content and area% calcification were measured to evaluate the effect of CaMg. To study the effect of CaMg on bone remodeling, rats underwent 5/6th nephrectomy combined with either a normal phosphorus diet or a high phosphorus diet to differentiate between possible bone effects resulting from either CaMg-induced phosphate deficiency or a direct effect of Mg. Vehicle or CaMg was administered at doses of 185 and 375 mg/kg/day for 8 weeks. Bone histomorphometry was performed. Aortic calcium content was significantly reduced by 185 mg/kg/day CaMg. CaMg ameliorated features of hyperparathyroid bone disease. In CRF rats on a normal phosphorus diet, the highest CaMg dose caused an increase in osteoid area due to phosphate depletion. The high phosphorus diet combined with the highest CaMg dose prevented the phosphate depletion and thus the rise in osteoid area. CaMg had no effect on osteoblast/osteoclast or dynamic bone parameters, and did not alter bone Mg levels. CaMg at doses that reduce vascular calcification did not show any harmful effect on bone turnover

Urinary phosphorus levels in 5/6^th nephrectomized rats on a normal (NP) or high phosphorus (HP) diet treated with either vehicle (veh), 185 or 375 mg/kg/day CaMg.

<p>*: p<0.05 vs NP, same dose; #: p<0.05 vs vehicle, same diet; °: p<0.05 vs week −4, same diet and dose.</p

Setup of the study addressing the effect of calcium acetate/magnesium carbonate (CaMg) on bone.

<p>The three groups presented in the clear bars received a normal phosphorus diet; those presented in the hatched bars received a high phoshorus diet. Nx: 5/6^th nephrectomy.</p

Calcium and magnesium content in the tibia of 5/6^th nephrectomized rats on a normal (NP) or high phosphorus (HP) diet treated with either vehicle (veh), 185 or 375 mg/kg/day CaMg.

<p>Calcium and magnesium content in the tibia of 5/6^th nephrectomized rats on a normal (NP) or high phosphorus (HP) diet treated with either vehicle (veh), 185 or 375 mg/kg/day CaMg.</p