Ayyalasomayajula, Savita

Abstract Rationale: While survival of premature infants has improved with NICU interventions, prematurity remains a risk for developing chronic lung diseases. Oxygen supplementation in the NICU is unfortunately necessary, and clinical practice has shifted towards use of moderate hyperoxia (<60% O2) to minimize detrimental effects of very high O2 concentration (80-90%) such as bronchopulmonary dysplasia. However, even moderate hyperoxia increases the risk of bronchial airway diseases and wheezing/asthma with airway hyperreactivity and remodeling. We previously found that moderate hyperoxia induces cellular senescence in human fetal airway smooth muscle cells (fASM) and fibroblasts. However, the mechanisms by which hyperoxia enhances cellular senescence in fASM are not known. We investigated the role of endoplasmic reticulum (ER) stress and mitochondrial dysfunction, using fASM cells exposed to 21% (normoxia) vs. 50% O2 (hyperoxia). Methods: fASM isolated from lungs of 18-20 week aborted fetuses (Mayo IRB exempt; period of rapid bronchial airway growth) were exposed to normoxia, hyperoxia or etoposide (10 µM; positive control for senescence induction). Cells and lysates were harvested at days 2, 3 or 7 to assess markers of senescence markers, ER stress and mitochondrial morphology, and levels of cytosolic and mitochondrial ROS, using real-time fluorescence imaging, Western blotting, immunofluorescence, and a novel immuno-FISH staining of telomere associated foci (TAF). Mitochondria-specific antioxidant MitoQ [100 nM], ER stress inhibitor Salubrinal [12.5 µM] or mitochondrial fission inhibitor Mdivi-1 [10 µM] were used to assess their effects on senescence markers and interactions between ER stress and mitochondria in inducing senescence. Results: Hyperoxia enhanced cellular senescence via upregulation of p21, PAI-1, and Cav-1; downregulation of LMNB1 and Ki-67, with increased γH2A.X- and TAF-positive cells. Hyperoxia increased cytosolic and mitochondrial ROS, and markers of the three ER stress pathways (ATF6, IRE1α, XBP1s, PDI, GADD34). Hyperoxia altered mitochondrial morphology with increased fission protein Drp1 and fusion proteins Mfn1/2. Inhibition of Drp1 with Mdivi-1 blocked mitochondrial fragmentation, but not senescence. However, hyperoxia-induced p21 was reduced by the PERK pathway inhibitor Salubrinal and MitoQ. Conclusions: Hyperoxia induces senescence in fASM via mitochondrial ROS and ER stress: pathways that offer new opportunities to blunt detrimental effects of oxygen in developing lung via mitochondrial targeted antioxidants and/or ER stress inhibition.

Supplemental O 2 (hyperoxia) in premature infants detrimentally affects bronchial airways leading to increased senescence. Understanding the mechanisms by which hyperoxia initiates senescence in developing airways is critical for future therapeutic strategies. The current study showed that hyperoxia-induced senescence is mediated through increased mitochondrial reactive oxygen species and endoplasmic reticulum (ER) stress. ER stress inhibitors or mitochondria-targeted antioxidants may represent future therapies to blunt detrimental effects of supplemental oxygen in developing lungs.

ABSTRACT Background and Objective Previously, the novel small molecule ISFP10 has been shown to inhibit fungal phosphoglucomutase (PGM) activity in Aspergillus fumigatus and Pneumocystis spp. With 50-fold selectivity over the human PGM molecule due to the presence of a unique yet conserved cysteine residue present in a number pathogenic fungal PGMs, use of this compound may provide a novel broad-spectrum approach to treating fungal infections. Accordingly, we sought to determine the tolerability in test animals receiving this compound, as well as the potential antifungal activity of ISFP10 on cultures of the common fungal pathogens Candida albicans and Candida glabrata . Methods C57BL6 mice received once daily intraperitoneal (IP) injections of 100 mL of vehicle control (DMSO) or ISFP10 at a concentration of 10 mg/kg. Body weights were recorded daily for 7 days of treatment. On the final day, mice were weighed and euthanized. Postmortem blood collection was conducted via cardiac puncture and distributed to EDTA and lithium heparin tubes for complete blood count (CBC) and comprehensive blood chemistry panels, respectively. Liver, kidney, and lung tissue were also harvested and placed in 10% formalin for H&E staining and blinded histopathologic scoring. Lung samples were further analyzed for proinflammatory cytokines using enzyme-linked immunosorbent assays (ELISA) and quantitative PCR (qPCR). Furthermore, ISPF10 was tested for antifungal activity via 8-hour growth curve analysis in a concentration-dependent fashion against Candida albicans and Candida glabrata . Results There was no significant difference in the daily or final body weights of the mice receiving 10 mg/kg of ISFP10 compared to those of the vehicle control group. Extracellular matrix (ECM) transcripts for IL-6 and TNFα were statistically similar via qPCR. ELISA results of proinflammatory cytokines for IL-6 was not significant whereas TNFα levels in lung tissue from the ISFP10 treatment group were significantly reduced, indicating a potential anti-inflammatory effect of ISFP10 at this dosage. Overall, blood chemistry and CBC analysis revealed no overall significant differences between the two groups, except for increased neutrophil counts and decreased potassium levels in samples collected from ISFP10 treated animals compared to the vehicle control group. These laboratory abnormalities were not of clinical significance to the test animals. Blinded histopathological examination revealed no abnormalities or evidence of critical organ toxicity from all groups. Inhibition of C. albicans and C. glabrata culture growth by ISFP10 was concentration-dependent in YPD liquid media containing the ISFP10 compared to vehicle control. Conclusions Our preliminary testing of ISFP10 revealed no inherent safety or toxicology concerns within the observed parameters. These data further support significant culture suppressive activity against C. albicans and C. glabrata . Taken together, these observations of ISFP10 further indicate that targeting PGM might be a novel and viable therapeutic strategy for serious fungal infections. Key points An inhibitor specific to fungal PGM enzymes, termed ISFP10, was generally well tolerated when administered via intraperitoneal (IP) injection in mice. ISFP10 displays antifungal activity in a concentration-dependent manner against C. albicans and C. glabrata .

Our previous study suggests that glial-derived neurotrophic factor (GDNF) is involved in allergen-induced airway hyperreactivity and remodeling in vivo. Here, we show that GDNF has no direct effects in remodeling of human airway smooth muscle (ASM) but GDNF dysregulates mitochondrial morphology in human ASM in the context of asthma.