Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.

BACKGROUND: Although cardiac c-kit+ cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit+ cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit+ cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit+ cells. METHODS: We used single-cell sequencing and genetic lineage tracing of c-kit+ cells to determine whether various pathological stimuli would result in different fates of c-kit+ cells. RESULTS: Single-cell sequencing of cardiac CD45-c-kit+ cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit+ cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit+ cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. CONCLUSIONS: These results demonstrate that different pathological stimuli induce different cell fates of c-kit+ cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit+ cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit+ cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit+ cells.

Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans.

Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow­derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte­derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms.

Changes in Polyphenols Contents and Antioxidant Capacities of Organically and Conventionally Cultivated Tomato (Solanum lycopersicum L.) Fruits during Ripening.

Polyphenols of fruits and vegetables form an important part of human dietary compounds. Relatively little is known about accumulation of phenolics during fruits ripening process. The goal of this work was to study the changes in antioxidant activity and in content of 30 polyphenols during ripening of tomato fruits. Five organically and conventionally grown tomato cultivars were investigated at three different ripening stages. Phenolic compounds were extracted with methanol and extracts were analyzed by HPLC-DAD-MS/MS. During ripening, four different changing patterns were observed: (1) high level in green fruits with minimal changes; (2) continuous increase with maximum level in red-ripe fruits; (3) decrease; (4) increase and achieving maximum level at half-ripe stage. Similar change patterns were found for organic and conventional fruits. The accumulation patterns of phenolic compounds were similar in standard-type tomatoes but differed in several cases in cherry-type cultivar. Although contents of some polyphenols decreased during ripening, total phenolics and free radical scavenging activity increased in all studied cultivars and in case of both cultivation modes. The changes in content of phenolic compounds during ripening were greatly influenced by cultivars, but cultivation mode had only minor impact on dynamics in polyphenols contents in tomato fruits.

Three-year comparative study of polyphenol contents and antioxidant capacities in fruits of tomato (Lycopersicon esculentum Mill.) cultivars grown under organic and conventional conditions.

In the present study, four tomato cultivars were grown under organic and conventional conditions in separate unheated greenhouses in three consecutive years. The objective was to assess the influence of the cultivation system on the content of individual polyphenols, total phenolics, and antioxidant capacity of tomatoes. The fruits were analyzed for total phenolic content by the Folin-Ciocalteau method and antioxidant capacity by the DPPH free radical scavenging assay. Individual phenolic compounds were analyzed using HPLC-DAD-MS/MS. Among 30 identified and quantified polyphenols, significantly higher contents of apigenin acetylhexoside, caffeic acid hexoside I, and phloretin dihexoside were found in all organic samples. The content of polyphenols was more dependent on year and cultivar than on cultivation conditions. Generally, the cultivation system had minor impact on polyphenols content, and only a few compounds were influenced by the mode of cultivation in all tested cultivars during all three years.

Determinants of the heightened activity of glucocorticoid receptor translational isoforms.

Translational isoforms of the glucocorticoid receptor α (GR-A, -B, -C1, -C2, -C3, -D1, -D2, and -D3) have distinct tissue distribution patterns and unique gene targets. The GR-C3 isoform-expressing cells are more sensitive to glucocorticoid killing than cells expressing other GRα isoforms and the GR-D isoform-expressing cells are resistant to glucocorticoid killing. Whereas a lack of activation function 1 (AF1) may underlie the reduced activity of the GR-D isoforms, it is not clear how the GR-C3 isoform has heightened activity. Mutation analyses and N-terminal tagging demonstrated that steric hindrance is probably the mechanism for the GR-A, -B, -C1, and -C2 isoforms to have lower activity than the GR-C3 isoform. In addition, truncation scanning analyses revealed that residues 98 to 115 are critical in the hyperactivity of the human GR-C3 isoform. Chimera constructs linking this critical fragment with the GAL4 DNA-binding domain showed that GR residues 98 to 115 do not contain any independent transactivation activity. Mutations at residues Asp101 or Gln106 and Gln107 all reduced the activity of the GR-C3 isoform. In addition, functional studies indicated that Asp101 is crucial for the GR-C3 isoform to recruit coregulators and to mediate glucocorticoid-induced apoptosis. Thus, charged and polar residues are essential components of an N-terminal motif that enhances the activity of AF1 and the GR-C3 isoform. These studies, together with the observations that GR isoforms have cell-specific expression patterns, provide a molecular basis for the tissue-specific functions of GR translational isoforms.