Roxadustat (FG4592) alleviates Radiation-Induced Intestinal Injury by facilitating recovery of intestinal stem cell and reducing damage of intestinal epithelial cell

Background: Radiation induced-intestinal injury (RIII) is a common complication after radiation therapy in patients with pelvic, abdominal or retroperitoneal tumors. The mechanism of RIII includes radiation-induced death of intestinal epithelial cells (IECs) and damage of intestinal stem cells (ISCs), among which damage of ISCs is main cause. Most recently, hypoxia Inducible factor (HIF) has been found to have effect on maintaining stemness and promoting proliferation of ISCs, which suggests a protective role of HIF in the RIII. In this study, we investigated the effect of FG4592, a novel up-regulator of HIF, on the protection of RIII, and further researched its function on ISCs.Methods: With/without FG4592 treatment, the abdomen of mice was radiated at dose of 25Gy, and then the degree of intestinal injury was assessed by H&E staining and Brdu label. By intestinal organoid culture, the multiplication capacity and differentiation features of ISCs were detected. Besides, the effects of FG4592 on the radiated IECs were also evaluated by detecting cell viability, apoptosis, and proliferation potential.Results: FG4592 could effectively up-regulate the expression of HIF-1α and HIF-2α in vivo. An abdominal radiation of 25Gy established the RIII model of mice, by which FG4592 was verified to have protective effect on the intestine from radiation. Morphology and Brdu test of intestinal organoid showed that FG4592 could promote regeneration and differentiation in ISCs after radiation, which were mediated by up-regulating HIF-2 rather than HIF-1.Conclusion: FG4592, a novel up-regulator of HIF could remit RIII and promote regeneration and differentiation of ISCs after radiation, which were depend on HIF-2 rather than HIF-1.

Phenotypic Analysis of BrdU Label-Retaining Cells during the Maturation of Conducting Airway Epithelium in a Porcine Lung

Stem/progenitor cells have recently been demonstrated to play key roles in the maturation, injury repair, and regeneration of distinct organs or tissues. Porcine has spurred an increased interest in biomedical research models and xenotransplantation, owing to most of its organs share similarities in physiology, cellular composition and size to humans. Therefore, characterization of stem/progenitor cells in porcine organs or tissues may provide a novel avenue to better understand the biology and function of stem cells in humans. In the present study, potential stem/progenitor cells in conducting airway epithelium of a porcine lung were characterized by morphometric analysis of bromodeoxyuridine (BrdU) label-retaining cells (LRCs) during the maturation of the lung. The results showed a pseudostratified mucociliary epithelium comprised of basal, ciliated, goblet, and columnar cells in the conducting airway of a porcine lung. In addition, the majority of primary epithelial cells able to proliferate in vitro expressed keratin 5, a subpopulation of these keratin 5-positive cells, also expressed CD117 (c-Kit) or CD49f (integrin alpha 6, ITGA6), implying that they might be potential epithelial stem/progenitor cells in conducting airway of a porcine lung. Lineage tracing analysis with a BrdU-labeled neonatal piglet showed that the proportion of BrdU-labeled cells in conducting airways decreased over the 90-day period of lung maturation. The BrdU-labeled epithelial cells also expressed keratin 14, mucin 5AC, or prosurfactant protein C (ProSP-C); among them, the keratin 14-positive cells were the most frequent BrdU-labeled epithelial cell type as determined by immunohistochemical and immunofluorescence staining. This study may provide valuable information on the biology and function of epithelial stem/progenitor cells in conducting airway of pigs and humans.

Abstract 20: Caridomoyocyte Biology Revealed by Fluorescence Ubiquitination-based Cell-cycle Indicators

Existing myocyte contribution to new myocyte formation remains an active area of investigation. Novel experimental methodology is needed to faithfully label cardiomyocyte cell-cycle activity after birth and following injury. The Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) system can be used to aid visualization of cell cycle activity and progression by monitoring the inverse oscillation dynamics of fluorescently tagged cell cycle fusion proteins AzG-hGeminin and mKO2-hCdt1. Using this system, we hypothesize that cardiomyocytes retain the capacity to cycle throughout postnatal development and re-enter the cell cycle following acute myocardial infarction injury (MI). A novel cardiac specific FUCCI transgenic mouse model, αMHC-FUCCI, was developed to study cell-cycle dynamics of cardiomyocytes. αMHC-FUCCI hearts were collected throughout postnatal (PN) development to examine cardiomyocyte cell-cycle. Similarly, adult αMHC-FUCCI mice were subjected to MI, injected daily with BrdU and harvested at 3, 7, 10, 14 and 21 days post-MI for further analysis. Peak incidence of single mKO2-hCdt1 (7%, G 1 ) and AzG-hGem (2%, S/G 2 /M) fluorescence in cardiomyocytes occurs at PN7 and decreases over time as as confirmed by colocalization with BrdU and/or mitotic marker phospho-histone 3. Interestingly, continued mitotic activity exists at PN14 as observed by AzG+/pH3+ myocytes and concurrent mKO2+/AzG+ fluorescence is observed in 60% of adult myocardium by at one month. Together, these results indicate cardiac myocytes remain active at least two weeks after birth and transition into a G1/S phase as opposed to a mitotic exit (G0) as adults. Intriguingly, BrdU+ label is only detected in the non-myocyte interstitial population in and around the border zone through the first two weeks post-MI. BrdU+/AzG+ and or/mKO+ myocytes are detectable at 21days post-MI, indicating a lag in cardiomyocyte cell cycle re-entry. These results suggest myocytes retain the ability to re-enter the cell cycle at low levels three weeks post-MI. Future studies will analyze cardiomyocyte cell-cycle biology in response to diffuse injury and will further elucidate the mechanism behind myocardial regeneration.

ID: 90: ADAM10 REGULATES STEMNESS OF ADULT NEURAL STEM CELLS BY MEDIATING THEIR POSITIONING WITHIN THE SVZ NICHE

The subventricular zone (SVZ) niche has been defined to have two distinct microenivroments, an apical compartment that is directly in contact with CSF and a basal compartment with a rich vascular network. Both these compartments regulate neural stem cell (NSC) properties via a diverse array of extrinsic cues by regulating both NSC-NSC and NSC-niche interactions. A majority of these signaling mechanisms are initiated by metalloproteinases. In this study, we investigated one such molecule, a disintegrin and metalloproteinase 10 (ADAM10) and its role in maintaining NSC properties. Using a conditional knockout, in which ADAM10 is specifically deleted in NSCs and NPCs (Nestin-CRE™/ADAM10fl/fl; denoted as KO) we noted altered contacts within the apical and basal compartments. BrdU pulse-chase studies demonstrated an increased number of long-term retaining BrdU NSCs in these KO mice. We also observed decreased BrdU label retaining neurons in the olfactory bulb in addition to a decreased neuroblast population in the SVZ indicating impaired neurogenesis in the KO mice. Intriguingly, we also observed cytoskeletal changes in NSCs and decreased migration of cells from the core of neurospheres derived from KO SVZ cells. This altered cell shape could intrinsically affect cell cycle progression. Taken together, this data suggests ADAM10 activity within the SVZ niche regulates NSC intrinsic properties and alters their cell cycle progression. Moreover, ADAM10 deletion appears to promote an aberrant self-renewal phenotype in NSCs preventing their differentiation. A more thorough understanding of how ADAM10 mediates these cellular changes and the underlying molecular mechanisms could be crucial in enhancing SVZ neurogenesis.