PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

The smoking-associated oxidant hypothiocyanous acid induces endothelial nitric oxide synthase dysfunction.

Smokers have an elevated risk of cardiovascular disease but the origin(s) of this increased risk are incompletely defined. Considerable evidence supports an accumulation of the oxidant-generating enzyme MPO (myeloperoxidase) in the inflamed artery wall, and smokers have high levels of SCN(-), a preferred MPO substrate, with this resulting in HOSCN (hypothiocyanous acid) formation. We hypothesized that this thiol-specific oxidant may target the Zn(2+)-thiol cluster of eNOS (endothelial nitric oxide synthase), resulting in enzyme dysfunction and reduced formation of the critical signalling molecule NO•. Decreased NO• bioavailability is an early and critical event in atherogenesis, and HOSCN-mediated damage to eNOS may contribute to smoking-associated disease. In the present study it is shown that exposure of isolated eNOS to HOSCN or MPO/H2O2/SCN(-) decreased active dimeric eNOS levels, and increased inactive monomer and Zn(2+) release, compared with controls, HOCl (hypochlorous acid)- or MPO/H2O2/Cl(-)-treated samples. eNOS activity was increasingly compromised by MPO/H2O2/Cl(-) with increasing SCN(-) concentrations. Exposure of HCAEC (human coronary artery endothelial cell) lysates to pre-formed HOSCN, or MPO/H2O2/Cl(-) with increasing SCN(-), increased eNOS monomerization and Zn(2+) release, and decreased activity. Intact HCAECs exposed to HOCl and HOSCN had decreased eNOS activity and NO2(-)/NO3(-) formation (products of NO• decomposition), and increased free Zn(2+). Exposure of isolated rat aortic rings to HOSCN resulted in thiol loss, and decreased eNOS activity and cGMP levels. Overall these data indicate that high SCN(-) levels, as seen in smokers, can increase HOSCN formation and enhance eNOS dysfunction in human endothelial cells, with this potentially contributing to increased atherogenesis in smokers.

Cardio-protective signalling by glyceryl trinitrate and cariporide in a model of donor heart preservation.

BACKGROUND: Storage of donor hearts in cardioplegic solutions supplemented with agents that mimic the ischaemic preconditioning response enhanced their post-reperfusion function. The present study examines the minimisation of cell death and activation of pro-survival signalling directed towards maintenance of mitochondrial homeostasis in hearts arrested and stored in two such agents, glyceryl-trinitrate, a nitric oxide donor and cariporide, (a sodium-hydrogen exchange inhibitor). METHODS: After baseline functional measurement, isolated working rat hearts were arrested and stored for 6h at 4°C in either Celsior(®), Celsior(®) containing 0.1mg/ml glyceryl-trinitrate, 10µM cariporide or both agents. After reperfusion, function was remeasured. Hearts were then processed for immunoblotting or histology. RESULTS: Necrotic and apoptotic markers present in the Celsior(®) group post-reperfusion were abolished by glyceryl-trinitrate, cariporide or both. Increased phosphorylation of ERK and Bcl2, after reperfusion in groups stored in glyceryl-trinitrate, cariporide or both along with increased phospho-STAT3 levels in the glyceryl-trinitrate/cariporide group correlated with functional recovery. Inhibition of STAT3 phosphorylation blocked recovery. No phospho-Akt increase was seen in any treatment. CONCLUSIONS: Activation of signalling pathways that favour mitophagy activation (ERK and Bcl2 phosphorylation) and maintenance of mitochondrial transition pore closure after reperfusion (STAT3 and ERK phosphorylation) were crucial for functional recovery of the donor heart.

Improved poststorage cardiac function by poly (ADP-ribose) polymerase inhibition: role of phosphatidylinositol 3-kinase Akt pathway.

BACKGROUND: Inhibition of poly(ADP-ribose) polymerase 1 (PARP) has been shown to be effective in minimizing cardiac ischemia reperfusion injury. We investigated the cardioprotective effect of the PARP inhibitor, INO-1153, in isolated working rat hearts after 6 hr of hypothermic storage in Celsior. METHODS: Hearts were treated with 1 muM INO-1153 before hypothermic storage, at cardioplegia and storage or after hypothermic storage. Hearts not exposed to INO-1153 served as controls. Another group was pretreated with the phosphatidylinositol 3-kinase inhibitor Wortmannin (0.1 muM) before storage in INO-1153-supplemented Celsior. After baseline measurement of aortic flow, heart rate, coronary flow, and cardiac output were obtained, hearts were arrested and stored in Celsior at 2-3 degrees C for 6 hr. After storage, hearts were reperfused for 15 min before performing work for a further 30 min, at which time poststorage indices of cardiac function were remeasured then heart tissue was stored at -80 degrees C for Western blot analysis. RESULTS: The presence of INO-1153 during prestorage perfusion or during cardioplegia and storage significantly improved poststorage cardiac function. Functional improvements produced by INO-1153 were completely abolished by Wortmnanin pretreatment. Western blots showed a significant increase in phospho-Akt in presence of INO-1153, which was inhibited by Wortmannin. CONCLUSION: Activation of the prosurvival phosphatidylinositol 3-kinase-Akt pathway was involved in the protective action of PARP inhibition in this model of donor heart procurement and hypothermic storage. Importantly for the logistics of clinical organ procurement, maximum protection is observed when the PARP inhibitor is included in the cardioplegic storage solution.

A recombinant human neuregulin-1 peptide improves preservation of the rodent heart after prolonged hypothermic storage.

BACKGROUND: Donor hearts are subjected to ischemia-reperfusion injury during transplantation. Recombinant human neuregulin (rhNRG)-1 peptide attenuates myocardial injury in various animal models of cardiomyopathy. Supplementing the organ-storage solution, Celsior (C), with glyceryl trinitrate (GTN) and cariporide improves cardiac preservation after hypothermic storage. We hypothesized that the addition of rhNRG-1 to C would improve cardiac preservation after hypothermic storage and provide incremental benefit in combination with GTN and cariporide. METHODS: An isolated working rat heart model was used. To assess the effect of rhNRG-1, hearts were stored for 6 hr at 4°C in C ± rhNRG-1 (14 nM). To assess the effect of using a combination of prosurvival kinase activators on cardiac preservation, the ischemic storage time was extended to 10 hr and hearts stored in C ± rhNRG-1 (14 nM) ± GTN (0.1 mg/mL) ± Cariporide (10 µM). Hearts were subsequently reperfused, cardiac function remeasured, and tissue collected for protein analysis and immunohistochemistry. Optimal timing of rhNRG-1 administration was also assessed. RESULTS: rhNRG-1 supplemented C improved functional recovery after 6 hr of storage (cardiac output recovery [mean ± SEM]: control 1.4% ± 0.6%; rhNRG-1+C 21.1% ± 7.9%; P<0.05). After 10-hr storage, no improvement in functional recovery was observed with rhNRG-1, GTN, or cariporide alone; however, GTN combined with cariporide did improve recovery (P<0.01), which was further enhanced by the addition of rhNRG-1 (P<0.01). Functional improvements were accompanied by increased phosphorylation of Akt, ERK1/2, STAT3, and GSK-3ß and reduced cleaved caspase-3 (P<0.01). CONCLUSIONS: rhNRG-1 given together with other activators of prosurvival pathways improves preservation of the rat heart and shows promise for increasing the cold-ischemic life of donor hearts in transplantation.

AIF suppresses chemical stress-induced apoptosis and maintains the transformed state of tumor cells.

Apoptosis-inducing factor (AIF) exhibits reactive oxygen species (ROS)-generating NADH oxidase activity of unknown significance, which is dispensable for apoptosis. We knocked out the aif gene in two human colon carcinoma cell lines that displayed lower mitochondrial complex I oxidoreductase activity and produced less ROS, but showed increased sensitivity to peroxide- or drug-induced apoptosis. AIF knockout cells failed to form tumors in athymic mice or grow in soft agar. Only AIF with intact NADH oxidase activity restored complex I activity and anchorage-independent growth of aif knockout cells, and induced aif-transfected mouse NIH3T3 cells to form foci. AIF knockdown in different carcinoma cell types resulted in lower superoxide levels, enhanced apoptosis sensitivity and loss of tumorigenicity. Antioxidants sensitized AIF-expressing cells to apoptosis, but had no effect on tumorigenicity. In summary, AIF-mediated resistance to chemical stress involves ROS and probably also mitochondrial complex I. AIF maintains the transformed state of colon cancer cells through its NADH oxidase activity, by mechanisms that involve complex I function. On both counts, AIF represents a novel type of cancer drug target.