Methionine adenosyltransferase2A inhibition restores metabolism to improve regenerative capacity and strength of aged skeletal muscle.

We investigate the age-related metabolic changes that occur in aged and rejuvenated myoblasts using in vitro and in vivo models of aging. Metabolic and signaling experiments reveal that human senescent myoblasts and myoblasts from a mouse model of premature aging suffer from impaired glycolysis, insulin resistance, and generate Adenosine triphosphate by catabolizing methionine via a methionine adenosyl-transferase 2A-dependant mechanism, producing significant levels of ammonium that may further contribute to cellular senescence. Expression of the pluripotency factor NANOG downregulates methionine adenosyltransferase 2 A, decreases ammonium, restores insulin sensitivity, increases glucose uptake, and enhances muscle regeneration post-injury. Similarly, selective inhibition of methionine adenosyltransferase 2 A activates Akt2 signaling, repairs pyruvate kinase, restores glycolysis, and enhances regeneration, which leads to significant enhancement of muscle strength in a mouse model of premature aging. Collectively, our investigation indicates that inhibiting methionine metabolism may restore age-associated impairments with significant gain in muscle function.

Reversine ameliorates hallmarks of cellular senescence in human skeletal myoblasts via reactivation of autophagy.

Cellular senescence leads to the depletion of myogenic progenitors and decreased regenerative capacity. We show that the small molecule 2,6-disubstituted purine, reversine, can improve some well-known hallmarks of cellular aging in senescent myoblast cells. Reversine reactivated autophagy and insulin signaling pathway via upregulation of Adenosine Monophosphate-activated protein kinase (AMPK) and Akt2, restoring insulin sensitivity and glucose uptake in senescent cells. Reversine also restored the loss of connectivity of glycolysis to the TCA cycle, thus restoring dysfunctional mitochondria and the impaired myogenic differentiation potential of senescent myoblasts. Altogether, our data suggest that cellular senescence can be reversed by treatment with a single small molecule without employing genetic reprogramming technologies.

Well-Defined pH-Responsive Self-Assembled Block Copolymers for the Effective Codelivery of Doxorubicin and Antisense Oligonucleotide to Breast Cancer Cells.

The worldwide steady increase in the number of cancer patients motivates the development of innovative drug delivery systems for combination therapy as an effective clinical modality for cancer treatment. Here, we explored a design concept based on poly(ethylene glycol)-b-poly(2-(dimethylamino)ethyl methacrylate)-b-poly(2-hydroxyethyl methacrylate-formylbenzoic acid) [PEG-b-PDMAEMA-b-P(HEMA-FBA)] for the dual delivery of doxorubicin (DOX) and GTI2040 (an antisense oligonucleotide for ribonucleotide reductase inhibition) to MCF-7 breast cancer cells. PEG-b-PDMAEMA-b-PHEMA, the precursor copolymer, was prepared through chain extensions from a PEG-based macroinitiator via two consecutive atom transfer radical polymerization (ATRP) steps. Then, it was modified at the PHEMA block with 4-formylbenzoic acid (FBA) to install reactive aldehyde moieties. A pH-responsive polymer-drug conjugate (PDC) was obtained by conjugating DOX to the polymer structure via acid-labile imine linkages, and subsequently self-assembled in an aqueous solution to form DOX-loaded self-assembled nanoparticles (DOX-SAN) with a positively charged shell. DOX-SAN condensed readily with negatively charged GTI2040 to form GTI2040/DOX-SAN nanocomplexes. Gel-retardation assay confirmed the affinity between GTI2040 and DOX-SAN. The GTI2040/DOX-SAN nanocomplex at N/P ratio of 30 exhibited a volume-average hydrodynamic size of 136.4 nm and a zeta potential of 21.0 mV. The pH-sensitivity of DOX-SAN was confirmed by the DOX release study based on the significant cumulative DOX release at pH 5.5 relative to pH 7.4. Cellular uptake study demonstrated favorable accumulation of GTI2040/DOX-SAN inside MCF-7 cells compared with free GTI2040/DOX. In vitro cytotoxicity study indicated higher therapeutic efficacy of GTI2040/DOX-SAN relative to DOX-SAN alone because of the downregulation of the R2 protein of ribonucleotide reductase. These outcomes suggest that the self-assembled pH-responsive triblock copolymer is a promising platform for combination therapy, which may be more effective in combating cancer than individual therapies.

NANOG Restores Contractility of Mesenchymal Stem Cell-Based Senescent Microtissues.

Mesenchymal stem cells (MSCs) have been extensively used in the field of tissue engineering as a source of smooth muscle cells (SMCs). However, recent studies showed deficits in the contractile function of SMCs derived from senescent MSCs and there are no available strategies to restore the contractile function that is impaired due to cellular or organismal senescence. In this study, we developed a tetracycline-regulatable system and employed micropost tissue arrays to evaluate the effects of the embryonic transcription factor, NANOG, on the contractility of senescent MSCs. Using this system, we show that expression of NANOG fortified the actin cytoskeleton and restored contractile function that was impaired in senescent MSCs. NANOG increased the expression of smooth muscle α-actin (ACTA2) as well as the contractile force generated by cells in three-dimensional microtissues. Interestingly, NANOG worked together with transforming growth factor-beta1 to further enhance the contractility of senescent microtissues. The effect of NANOG on contractile function was sustained for about 10 days after termination of its expression. Our results show that NANOG could reverse the effects of stem cell senescence and restore the myogenic differentiation potential of senescent MSCs. These findings may enable development of novel strategies to restore the function of senescent cardiovascular and other SMC-containing tissues.

NANOG Reverses the Myogenic Differentiation Potential of Senescent Stem Cells by Restoring ACTIN Filamentous Organization and SRF-Dependent Gene Expression.

Cellular senescence as a result of organismal aging or progeroid diseases leads to stem cell pool exhaustion hindering tissue regeneration and contributing to the progression of age related disorders. Here we discovered that ectopic expression of the pluripotent factor NANOG in senescent or progeroid myogenic progenitors reversed cellular aging and restored completely the ability to generate contractile force. To elicit its effects, NANOG enabled reactivation of the ROCK and Transforming Growth Factor (TGF)-ß pathways-both of which were impaired in senescent cells-leading to ACTIN polymerization, MRTF-A translocation into the nucleus and serum response factor (SRF)-dependent myogenic gene expression. Collectively our data reveal that cellular senescence can be reversed and provide a novel strategy to regain the lost function of aged stem cells without reprogramming to the pluripotent state. Stem Cells 2017;35:207-221.

Capture of endothelial cells under flow using immobilized vascular endothelial growth factor.

We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a surface consisting of heparin bound to poly-l-lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm(2)) to physiological (15 dyne/cm(2)). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC.

3D conductive nanocomposite scaffold for bone tissue engineering.

Bone healing can be significantly expedited by applying electrical stimuli in the injured region. Therefore, a three-dimensional (3D) ceramic conductive tissue engineering scaffold for large bone defects that can locally deliver the electrical stimuli is highly desired. In the present study, 3D conductive scaffolds were prepared by employing a biocompatible conductive polymer, ie, poly(3,4-ethylenedioxythiophene) poly(4-styrene sulfonate) (PEDOT:PSS), in the optimized nanocomposite of gelatin and bioactive glass. For in vitro analysis, adult human mesenchymal stem cells were seeded in the scaffolds. Material characterizations using hydrogen-1 nuclear magnetic resonance, in vitro degradation, as well as thermal and mechanical analysis showed that incorporation of PEDOT:PSS increased the physiochemical stability of the composite, resulting in improved mechanical properties and biodegradation resistance. The outcomes indicate that PEDOT:PSS and polypeptide chains have close interaction, most likely by forming salt bridges between arginine side chains and sulfonate groups. The morphology of the scaffolds and cultured human mesenchymal stem cells were observed and analyzed via scanning electron microscope, micro-computed tomography, and confocal fluorescent microscope. Increasing the concentration of the conductive polymer in the scaffold enhanced the cell viability, indicating the improved microstructure of the scaffolds or boosted electrical signaling among cells. These results show that these conductive scaffolds are not only structurally more favorable for bone tissue engineering, but also can be a step forward in combining the tissue engineering techniques with the method of enhancing the bone healing by electrical stimuli.