Vascular calcification is commonly seen in elderly people, though it can also appear in middle-aged subjects affected by premature vascular aging. The aim of this work is to test the involvement of microvesicles (MVs) produced by senescent endothelial cells (EC) and from plasma of elderly people in vascular calcification. The present work shows that MVs produced by senescent cultured ECs, plus those found in the plasma of elderly subjects, promote calcification in vascular smooth muscle cells. Only MVs from senescent ECs, and from elderly subjects' plasma, induced calcification. This ability correlated with these types of MVs' carriage of: a) increased quantities of annexins (which might act as nucleation sites for calcification), b) increased quantities of bone-morphogenic protein, and c) larger Ca contents. The MVs of senescent, cultured ECs, and those present in the plasma of elderly subjects, promote vascular calcification. The present results provide mechanistic insights into the observed increase in vascular calcification-related diseases in the elderly, and in younger patients with premature vascular aging, paving the way towards novel therapeutic strategies.

In mammalians, advancing age is associated with sarcopenia, the progressive and involuntary loss of muscle mass and strength. Hyperphosphatemia is an aging-related condition involved in several pathologies. The aim of this work was to assess whether hyperphosphatemia plays a role in the age-related loss of mass muscle and strength by inducing cellular senescence in murine myoblasts and to explore the intracellular mechanism involved in this effect. Cultured mouse C2C12 cells were treated with 10 mM beta-glycerophosphate (BGP] at different periods of time to induce hyperphosphatemia. BGP promoted cellular senescence after 24 h of treatment, assessed by the increased expression of p53, acetylated-p53 and p21 and senescence associated β-galactosidase activity. In parallel, BGP increased ILK expression and activity, followed by mTOR activation and autophagy reduction. Knocking-down ILK expression increased autophagy and protected cells from senescence induced by hyperphosphatemia. BGP also reduced the proliferative capacity of cultured myoblasts. Old mice (24-months-old] presented higher serum phosphate concentration, lower forelimb strength, higher expression of p53 and ILK and less autophagy in vastus muscle than young mice (5-months-old]. In conclusion, we propose that hyperphosphatemia induces senescence in cultured myoblasts through ILK overexpression, reducing their proliferative capacity, which could be a mechanism involved in the development of sarcopenia, since old mice showed loss of muscular strength correlated with high serum phosphate concentration and increased levels of ILK and p53.

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Background Hyperphosphatemia has been related to the development of sarcopenia in aging mice. We describe the intracellular mechanisms involved in the impairment of the myogenic differentiation promoted by hyperphosphatemia and analyse these mechanisms in the muscle from older mice. Methods C 2 C 12 cells were grown in 2% horse serum in order to promote myogenic differentiation, in the presence or absence of 10 mM beta-glycerophosphate (BGP) for 7 days. Troponin T, paired box 7 (Pax-7), myogenic factor 5 (Myf5), myogenic differentiation 1 (MyoD), myogenin (MyoG), myocyte enhancer factor 2 (MEF2C), P300/CBP-associated factor (PCAF), histone deacetylase 1 (HDAC1), fibronectin, vimentin, and collagen I were analysed at 48, 72, and 168 h, by western blotting or by immunofluorescence staining visualized by confocal microscopy. Studies in mice were performed in 5-and 24-month-old C57BL6 mice. Three months before sacrifice, 21-month-old mice were fed with a standard diet or a low phosphate diet, containing 0.6% or 0.2% phosphate, respectively. Serum phosphate concentration was assessed by a colorimetric method and forelimb strength by a grip test. Fibrosis was observed in the tibialis anterior muscle by Sirius Red staining. In gastrocnemius muscle, MyoG, MEF2C, and fibronectin expressions were analysed by western blotting. Results Cells differentiated in the presence of BGP showed near five times less expression of troponin T and kept higher levels of Pax-7 than control cells indicating a reduced myogenic differentiation. BGP reduced Myf5 about 50% and diminished MyoD transcriptional activity by increasing the expression of HDAC1 and reducing the expression of PCAF. Consequently, BGP reduced to 50% the expression of MyoG and MEF2C. A significant increase in the expression of fibrosis markers as collagen I, vimentin, and fibronectin was found in cells treated with BGP. In mice, serum phosphate (17.24 ± 0.77 mg/dL young; 23.23 ± 0.81 mg/dL old; 19.09 ± 0.75 mg/dL old with low phosphate diet) correlates negatively (r = À0.515, P = 0.001) with the muscular strength (3.13 ± 0.07 gf/g young; 1.70 ± 0.12 gf/ g old; 2.10 ± 0.09 gf/g old with low phosphate diet) and with the expression of MyoG (r = À0.535, P = 0.007) and positively with the expression of fibronectin (r = 0.503, P = 0.001) in gastrocnemius muscle. The tibialis anterior muscle from old mice showed muscular fibrosis. Older mice fed with a low phosphate diet showed improved muscular parameters relative to control mice of similar age. Conclusions Hyperphosphatemia impairs myogenic differentiation, by inhibiting the transcriptional activity of MyoD, and enhances the expression of fibrotic genes in cultured myoblasts. Experiments carried out in older mice demonstrate a close relationship between age-related hyperphosphatemia and the decrease in the expression of myogenic factors and the increase in factors related to muscle fibrosis.

The loss of muscular mass and strength is an inevitable aging related phenomenon, named sarcopenia. The mechanisms involved in its origin and progression remain to be clearly elucidated. Malnutrition, decreased physical exercise and the decline in sex steroid hormone production and in insulin sensitivity play an important role in sarcopenia [1]. Sarcopenic muscles suffer profound changes in their architecture, exhibiting both reduced fiber size and reduced fiber number, especially of the type II fibers, reduced number of satellite cells www.aging-us.com

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression.

Aging impairs vascular function, but the mechanisms involved are unknown. The aim of this study was to analyze whether aging-related hyperphosphatemia is implied in this effect by elucidating the role of oxidative stress. C57BL6 mice that were aged 5 months (young) and 24 months (old), receiving a standard (0.6%) or low-phosphate (0.2%) diet, were used. Isolated mesenteric arteries from old mice showed diminished endothelium-dependent vascular relaxation by the down-regulation of NOS3 expression, increased inflammation and increased fibrosis in isolated aortas, compared to those isolated from young mice. In parallel, increased Nox4 expression and reduced Nrf2, Sod2-Mn and Gpx1 were found in the aortas from old mice, resulting in oxidant/antioxidant imbalance. The low-phosphate diet improved vascular function and oxidant/antioxidant balance in old mice. Mechanisms were analyzed in endothelial (EC) and vascular smooth muscle cells (SMCs) treated with the phosphate donor ß-glycerophosphate (BGP). In EC, BGP increased Nox4 expression and ROS production, which reduced NOS3 expression via NFκB. BGP also increased inflammation in EC. In SMC, BGP increased Collagen I and fibronectin expression by priming ROS production and NFκB activity. In conclusion, hyperphosphatemia reduced endothelium-dependent vascular relaxation and increased inflammation and vascular fibrosis through an impairment of oxidant/antioxidant balance in old mice. A low-phosphate diet achieved improvements in the vascular function in old mice.

In chronic kidney disease, systemic inflammation and high serum phosphate (P) promote the de-differentiation of vascular smooth muscle cells (VSMC) to osteoblast-like cells, increasing the propensity for medial calcification and cardiovascular mortality. Vascular microRNA-145 (miR-145) content is essential to maintain VSMC contractile phenotype. Because vitamin D induces aortic miR-145, uremia and high serum P reduce it and miR-145 directly targets osteogenic osterix in osteoblasts, this study evaluated a potential causal link between vascular miR-145 reductions and osterix-driven osteogenic differentiation and its counter-regulation by vitamin D. Studies in aortic rings from normal rats and in the rat aortic VSMC line A7r5 exposed to calcifying conditions corroborated that miR-145 reductions were associated with decreases in contractile markers and increases in osteogenic differentiation and calcium (Ca) deposition. Furthermore, miR-145 silencing enhanced Ca deposition in A7r5 cells exposed to calcifying conditions, while miR-145 overexpression attenuated it, partly through increasing α-actin levels and reducing osterix-driven osteogenic differentiation. In mice, 14 weeks after the induction of renal mass reduction, both aortic miR-145 and α-actin mRNA decreased by 80% without significant elevations in osterix or Ca deposition. Vitamin D treatment from week 8 to 14 fully prevented the reductions in aortic miR-145 and attenuated by 50% the decreases in α-actin, despite uremia-induced hyperphosphatemia. In conclusion, vitamin D was able to prevent the reductions in aortic miR-145 and α-actin content induced by uremia, reducing the alterations in vascular contractility and osteogenic differentiation despite hyperphosphatemia.