Summary Deletion of the p66Shc gene results in lean and healthy mice, retards aging, and protects from aging-associated diseases, raising the question of why p66 Shc has been selected, and what is its physiological role. We have investigated survival and reproduction of p66 Shc ) ⁄ ) mice in a population living in a large outdoor enclosure for a year, subjected to food competition and exposed to winter temperatures. Under these conditions, deletion of p66Shc was strongly counterselected. Laboratory studies revealed that p66 Shc ) ⁄ ) mice have defects in fat accumulation, thermoregulation, and reproduction, suggesting that p66Shc has been evolutionarily selected because of its role in energy metabolism. These findings imply that the health impact of targeting aging genes might depend on the specific energetic niche and caution should be exercised against premature conclusions regarding gene functions that have only been observed in protected laboratory conditions.
In tissue engineering, survival of larger constructs remains challenging due to limited supply with oxygen caused by a lack of early vascularization. Controlled release of oxygen from small organic molecules represents a possible strategy to prevent cell death under anoxic conditions. A comprehensive study of methylated pyridone-derived endoperoxides has led to the development of water-soluble molecules that undergo retro Diels-Alder reactions in aqueous environment releasing oxygen in high yield and with half-lives of up to 13 hours. These molecules in combination with vitamin C as singlet oxygen quencher significantly improved survival of 3T3 fibroblasts and rat smooth muscle cells challenged with oxygendepleted conditions. ABSTRACT. In tissue engineering, survival of larger constructs remains challenging due to limited supply with oxygen caused by a lack of early vascularization. Controlled release of oxygen from small organic molecules represents a possible strategy to prevent cell death under anoxic conditions. A comprehensive study of methylated pyridone-derived endoperoxides has led to the development of water-soluble molecules that undergo retro Diels-Alder reactions in aqueous environment releasing oxygen in high yield and with half-lives of up to 13 hours. These molecules in combination with vitamin C as singlet oxygen quencher significantly improved survival of 3T3 fibroblasts and rat smooth muscle cells challenged with oxygen-depleted conditions.
The object of this study was to investigate the role of scaffold porosity on tissue ingrowth using hybrid scaffolds consisting of bladder acellular matrix and electrospun poly (lactide-co-glycolide) (PLGA) microfibers that mimic the morphological characteristics of the bladder wall in vitro and in vivo. We compared single-spun (SS) PLGA scaffolds with more porous cospun (CS) scaffolds (PLGA and polyethylene glycol). Scaffolds were characterized by scanning electron microscopy. Bladder smooth muscle cells (SMCs) were seeded, and proliferation and histological assays were performed. Sixteen rats were subjected to augmentation cystoplasty with seeded SS or CS scaffolds, morphological, and histological studies were performed 2 and 4 weeks after implantation. The porosities of SS and CS scaffolds were 73.1 ± 2.9% and 80.9 ± 1.5%, respectively. The in vitro evaluation revealed significantly deeper cell migration into CS scaffolds. The in vivo evaluation showed significant shrinkage of SS scaffolds (p = 0.019). The histological analysis revealed a bladder wall-like structure with urothelial lining and SMC infiltration in both groups. The microvessel density was significantly increased in the CS scaffolds (p < 0.001). Increasing the porosity of electrospun hybrid scaffolds is an effective strategy to enhance cell proliferation and distribution in vitro and tissue ingrowth in vivo.
Aging is accompanied by poor learning and memory abilities and by decreased hippocampal neurogenesis, a process that is also modulated by oxidative stress (OS). P66(Shc) has recently emerged as a novel mammalian gerontogene able to affect healthspan during aging. Deletion of this gene in mice leads to reduced OS accompanied by decreased incidence of age-related pathologies and reduced signs of behavioral aging. We hypothesized that p66(Shc-/-) mutants might show increased neurogenesis in the hippocampus, a brain region involved in learning and memory processes. To this aim, granule cell number, proliferation, neuronal differentiation, and cell death were assessed in the hippocampus in senescent p66(Shc-/-) [knock out (KO)] and p66(Shc+/+) [wild type (WT)] male and female mice. Spatial learning abilities and spontaneous activity were also investigated in a multifunctional behavioral system-IntelliCages. The behavioral analysis revealed that females in general perform better in spatial learning tasks, with genotype effects being apparent in the activity pattern only. Likewise, all females showed increased neuronal differentiation, whereas increased proliferation was found only in those belonging to the p66(Shc-/-) genotype, indicating that they might be protected from precursor cell loss. The number of dying cells was not affected by genotype or sex; however, all KO mice showed less granule cells than WT. Overall, our data suggest that hippocampal function is protected in the female gender at older age, an effect amplified by reduced OS in the p66(Shc-/-) mutant.
Background Major retinal degenerative diseases, including age-related macular degeneration, diabetic retinopathy and retinal detachment, are associated with a local decrease in oxygen availability causing the formation of hypoxic areas affecting the photoreceptor (PR) cells. Here, we addressed the underlying pathological mechanisms of PR degeneration by focusing on energy metabolism during chronic activation of hypoxia-inducible factors (HIFs) in rod PR. Methods We used two-photon laser scanning microscopy (TPLSM) of genetically encoded biosensors delivered by adeno-associated viruses (AAV) to determine lactate and glucose dynamics in PR and inner retinal cells. Retinal layer-specific proteomics, in situ enzymatic assays and immunofluorescence studies were used to analyse mitochondrial metabolism in rod PRs during chronic HIF activation. Results PRs exhibited remarkably higher glycolytic flux through the hexokinases than neurons of the inner retina. Chronic HIF activation in rods did not cause overt change in glucose dynamics but an increase in lactate production nonetheless. Furthermore, dysregulation of the oxidative phosphorylation pathway (OXPHOS) and tricarboxylic acid (TCA) cycle in rods with an activated hypoxic response decelerated cellular anabolism causing shortening of rod photoreceptor outer segments (OS) before onset of cell degeneration. Interestingly, rods with deficient OXPHOS but an intact TCA cycle did not exhibit these early signs of anabolic dysregulation and showed a slower course of degeneration. Conclusion Together, these data indicate an exceeding high glycolytic flux in rods and highlight the importance of mitochondrial metabolism and especially of the TCA cycle for PR survival in conditions of increased HIF activity.
Background: Major retinal degenerative diseases, including age-related macular degeneration, diabetic retinopathy and retinal detachment, are associated with a local decrease in oxygen availability causing the formation of hypoxic areas affecting the photoreceptor (PR) cells. Here, we addressed the underlying pathological mechanisms of PR degeneration by focusing on energy metabolism during chronic activation of hypoxia-inducible factors (HIFs) in rod PR. Methods: We used two-photon laser scanning microscopy (TPLSM) of genetically encoded biosensors delivered by adeno-associated viruses (AAV) to determine lactate and glucose dynamics in PR and inner retinal cells. Retinal layer-specific proteomics, in situ enzymatic assays and immunofluorescence studies were used to analyse mitochondrial metabolism in rod PRs during chronic HIF activation. Results: PRs exhibited remarkably higher glycolytic flux through the hexokinases than neurons of the inner retina. Chronic HIF activation in rods did not cause overt change in glucose dynamics but an increase in lactate production nonetheless. Furthermore, dysregulation of the oxidative phosphorylation pathway (OXPHOS) and tricarboxylic acid (TCA) cycle in rods with an activated hypoxic response decelerated cellular anabolism causing shortening of rod photoreceptor outer segments (OS) before onset of cell degeneration. Interestingly, rods with deficient OXPHOS but an intact TCA cycle did not exhibit these early signs of anabolic dysregulation and showed a slower course of degeneration. Conclusion: Together, these data indicate an exceeding high glycolytic flux in rods and highlight the importance of mitochondrial metabolism and especially of the TCA cycle for PR survival in conditions of increased HIF activity.
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