Constantin Georgescu

IGF-1 has sexually dimorphic, pleiotropic, and time-dependent effects on healthspan, pathology, and lifespan

Reduced circulating levels of IGF-1 have been proposed as a conserved anti-aging mechanism that contributes to increased lifespan in diverse experimental models. However, IGF-1 has also been shown to be essential for normal development and the maintenance of tissue function late into the lifespan. These disparate findings suggest that IGF-1 may be a pleiotropic modulator of health and aging, as reductions in IGF-1 may be beneficial for one aspect of aging, but detrimental for another. We postulated that the effects of IGF-1 on tissue health and function in advanced age are dependent on the tissue, the sex of the animal, and the age at which IGF-1 is manipulated. In this study, we examined how alterations in IGF-1 levels at multiple stages of development and aging influence overall lifespan, healthspan, and pathology. Specifically, we investigated the effects of perinatal, post-pubertal, and late-adult onset IGF-1 deficiency using genetic and viral approaches in both male and female igff/f C57Bl/6 mice. Our results support the concept that IGF-1 levels early during lifespan establish the conditions necessary for subsequent healthspan and pathological changes that contribute to aging. Nevertheless, these changes are specific for each sex and tissue. Importantly, late-life IGF-1 deficiency (a time point relevant for human studies) reduces cancer risk but does not increase lifespan. Overall, our results indicate that the levels of IGF-1 during development influence late-life pathology, suggesting that IGF-1 is a developmental driver of healthspan, pathology, and lifespan.

Temporal and spatial regulation of epsin abundance and VEGFR3 signaling are required for lymphatic valve formation and function

Proteins involved in endocytosis promote the internalization and degradation of VEGFR3, ensuring valve formation in lymphatic vessels. Ensuring a One-Way Flow of Lymph Lymphatic vessels return lymph, which consists of fluid and mostly immune cells that go into the tissues from the blood, back to the circulation. Valves in lymphatic vessels ensure that lymph flows in one direction into veins and prevent the accumulation of lymph in tissues, a condition called edema. The receptor VEGFR3 is required for lymphatic vessel development. Liu et al. noted that the regions of developing lymphatic vessels that became valves had high VEGFR3 amounts but low amounts of epsin 1 and 2, proteins that are involved in endocytosis. They determined that epsin 1 and 2 suppressed VEGFR3 signaling in collecting lymphatic trunks by triggering endocytosis and degradation of VEGFR3. Furthermore, mice lacking epsin 1 and 2 in the endothelial cells that line lymphatic vessels had defective lymphatic valves and impaired drainage. Lymphatic valve formation was restored in these mice by deletion of a single allele of Vegfr3 or treatment with a VEGFR3 inhibitor. Lymphatic valves prevent the backflow of the lymph fluid and ensure proper lymphatic drainage throughout the body. Local accumulation of lymphatic fluid in tissues, a condition called lymphedema, is common in individuals with malformed lymphatic valves. The vascular endothelial growth factor receptor 3 (VEGFR3) is required for the development of lymphatic vascular system. The abundance of VEGFR3 in collecting lymphatic trunks is high before valve formation and, except at valve regions, decreases after valve formation. We found that in mesenteric lymphatics, the abundance of epsin 1 and 2, which are ubiquitin-binding adaptor proteins involved in endocytosis, was low at early stages of development. After lymphatic valve formation, the initiation of steady shear flow was associated with an increase in the abundance of epsin 1 and 2 in collecting lymphatic trunks, but not in valve regions. Epsin 1 and 2 bound to VEGFR3 and mediated the internalization and degradation of VEGFR3, resulting in termination of VEGFR3 signaling. Mice with lymphatic endothelial cell–specific deficiency of epsin 1 and 2 had dilated lymphatic capillaries, abnormally high VEGFR3 abundance in collecting lymphatics, immature lymphatic valves, and defective lymph drainage. Deletion of a single Vegfr3 allele or pharmacological suppression of VEGFR3 signaling restored normal lymphatic valve development and lymph drainage in epsin-deficient mice. Our findings establish a critical role for epsins in the temporal and spatial regulation of VEGFR3 abundance and signaling in collecting lymphatic trunks during lymphatic valve formation.

Complement inhibition decreases early fibrogenic events in the lung of septic baboons.

Acute respiratory distress syndrome (ARDS) induced by severe sepsis can trigger persistent inflammation and fibrosis. We have shown that experimental sepsis in baboons recapitulates ARDS progression in humans, including chronic inflammation and long‐lasting fibrosis in the lung. Complement activation products may contribute to the fibroproliferative response, suggesting that complement inhibitors are potential therapeutic agents. We have been suggested that treatment of septic baboons with compstatin, a C3 convertase inhibitor protects against ARDS‐induced fibroproliferation. Baboons challenged with 109 cfu/kg (LD50) live E. coli by intravenous infusion were treated or not with compstatin at the time of challenge or 5 hrs thereafter. Changes in the fibroproliferative response at 24 hrs post‐challenge were analysed at both transcript and protein levels. Gene expression analysis showed that sepsis induced fibrotic responses in the lung as early as 24 hrs post‐bacterial challenge. Immunochemical and biochemical analysis revealed enhanced collagen synthesis, induction of profibrotic factors and increased cell recruitment and proliferation. Specific inhibition of complement with compstatin down‐regulated sepsis‐induced fibrosis genes, including transforming growth factor‐beta (TGF‐β), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase 1 (TIMP1), various collagens and chemokines responsible for fibrocyte recruitment (e.g. chemokine (C‐C motif) ligand 2 (CCL2) and 12 (CCL12)). Compstatin decreased the accumulation of myofibroblasts and proliferating cells, reduced the production of fibrosis mediators (TGF‐β, phospho‐Smad‐2 and CTGF) and inhibited collagen deposition. Our data demonstrate that complement inhibition effectively attenuates collagen deposition and fibrotic responses in the lung after severe sepsis. Inhibiting complement could prove an attractive strategy for preventing sepsis‐induced fibrosis of the lung.

Chemokine receptors CXCR2 and CX3CR1 differentially regulate functional responses of bone-marrow endothelial progenitors during atherosclerotic plaque regression

AIMS Atherosclerosis manifests itself as arterial plaques, which lead to heart attacks or stroke. Treatments supporting plaque regression are therefore aggressively pursued. Studies conducted in models in which hypercholesterolaemia is reversible, such as the Reversa mouse model we have employed in the current studies, will be instrumental for the development of such interventions. Using this model, we have shown that advanced atherosclerosis regression occurs when lipid lowering is used in combination with bone-marrow endothelial progenitor cell (EPC) treatment. However, it remains unclear how EPCs home to regressing plaques and how they augment atherosclerosis reversal. Here we identify molecules that support functional responses of EPCs during plaque resolution. METHODS AND RESULTS Chemokines CXCL1 and CX3CL1 were detected in the vascular wall of atheroregressing Reversa mice, and their cognate receptors CXCR2 and CX3CR1 were observed on adoptively transferred EPCs in circulation. We tested whether CXCL1-CXCR2 and CX3CL1-CX3CR1 axes regulate functional responses of EPCs during plaque reversal. We show that pharmacological inhibition of CXCR2 or CX3CR1, or genetic inactivation of these two chemokine receptors interfered with EPC-mediated advanced atherosclerosis regression. We also demonstrate that CXCR2 directs EPCs to regressing plaques while CX3CR1 controls a paracrine function(s) of these cells. CONCLUSION CXCR2 and CX3CR1 differentially regulate EPC functional responses during atheroregression. Our study improves understanding of how chemokines and chemokine receptors regulate plaque resolution, which could determine the effectiveness of interventions reducing complications of atherosclerosis.

Effects of transgenic methionine sulfoxide reductase A (MsrA) expression on lifespan and age-dependent changes in metabolic function in mice

Mechanisms that preserve and maintain the cellular proteome are associated with long life and healthy aging. Oxidative damage is a significant contributor to perturbation of proteostasis and is dealt with by the cell through regulation of antioxidants, protein degradation, and repair of oxidized amino acids. Methionine sulfoxide reductase A (MsrA) repairs oxidation of free- and protein-bound methionine residues through enzymatic reduction and is found in both the cytosol and the mitochondria. Previous studies in Drosophila have shown that increasing expression of MsrA can extend longevity. Here we test the effects of increasing MsrA on longevity and healthy aging in two transgenic mouse models. We show that elevated expression of MsrA targeted specifically to the cytosol reduces the rate of age-related death in female mice when assessed by Gompertz analysis. However, neither cytosolic nor mitochondrial MsrA overexpression extends lifespan when measured by log-rank analysis. In mice with MsrA overexpression targeted to the mitochondria, we see evidence for improved insulin sensitivity in aged female mice. With these and our previous data, we conclude that the increasing MsrA expression in mice has differential effects on aging and healthy aging that are dependent on the target of its subcellular localization.

Use it or lose it: citations predict the continued online availability of published bioinformatics resources

Abstract Scientific Data Analysis Resources (SDARs) such as bioinformatics programs, web servers and databases are integral to modern science, but previous studies have shown that the Uniform Resource Locators (URLs) linking to them decay in a time-dependent manner, with ∼27% decayed to date. Because SDARs are overrepresented among sciences most cited papers over the past 20 years, loss of widely used SDARs could be particularly disruptive to scientific research. We identified URLs in MEDLINE abstracts and used crowdsourcing to identify which reported the creation of SDARs. We used the Internet Archives Wayback Machine to approximate ‘death dates’ and calculate citations/year over each SDARs lifespan. At first glance, decayed SDARs did not significantly differ from available SDARs in their average citations per year over their lifespan or journal impact factor (JIF). But the most cited SDARs were 94% likely to be relocated to another URL versus only 34% of uncited ones. Taking relocation into account, we find that citations are the strongest predictors of current online availability after time since publication, and JIF modestly predictive. This suggests that URL decay is a general, persistent phenomenon affecting all URLs, but the most useful/recognized SDARs are more likely to persist.

DNA replication timing during development anticipates transcriptional programs and parallels enhancer activation

In dividing cells, DNA replication occurs in a precise order, but many questions remain regarding the mechanisms of replication timing establishment and regulation. We now have generated genome-wide, high-resolution replication timing maps throughout zebrafish development. Unexpectedly, in the rapid cell cycles preceding the midblastula transition, a defined timing program was present that predicted the initial wave of zygotic transcription. Replication timing was thereafter progressively and continuously remodeled across the majority of the genome, and epigenetic changes involved in enhancer activation frequently paralleled developmental changes in replication timing. The long arm of Chromosome 4 underwent a dramatic developmentally regulated switch to late replication during gastrulation, reminiscent of mammalian X Chromosome inactivation. This study reveals that replication timing is dynamic and tightly linked to epigenetic and transcriptional changes throughout early zebrafish development. These data provide insight into the regulation and functions of replication timing and will enable further mechanistic studies.

Sexually divergent DNA methylation patterns with hippocampal aging

DNA methylation is a central regulator of genome function, and altered methylation patterns are indicative of biological aging and mortality. Age‐related cellular, biochemical, and molecular changes in the hippocampus lead to cognitive impairments and greater vulnerability to neurodegenerative disease that varies between the sexes. The role of hippocampal epigenomic changes with aging in these processes is unknown as no genome‐wide analyses of age‐related methylation changes have considered the factor of sex in a controlled animal model. High‐depth, genome‐wide bisulfite sequencing of young (3 month) and old (24 month) male and female mouse hippocampus revealed that while total genomic methylation amounts did not change with aging, specific sites in CG and non‐CG (CH) contexts demonstrated age‐related increases or decreases in methylation that were predominantly sexually divergent. Differential methylation with age for both CG and CH sites was enriched in intergenic and intronic regions and under‐represented in promoters, CG islands, and specific enhancer regions in both sexes, suggesting that certain genomic elements are especially labile with aging, even if the exact genomic loci altered are predominantly sex‐specific. Lifelong sex differences in autosomal methylation at CG and CH sites were also observed. The lack of genome‐wide hypomethylation, sexually divergent aging response, and autosomal sex differences at CG sites was confirmed in human data. These data reveal sex as a previously unappreciated central factor of hippocampal epigenomic changes with aging. In total, these data demonstrate an intricate regulation of DNA methylation with aging by sex, cytosine context, genomic location, and methylation level.

Trends in the production of scientific data analysis resources

BackgroundAs the amount of scientific data grows, peer-reviewed Scientific Data Analysis Resources (SDARs) such as published software programs, databases and web servers have had a strong impact on the productivity of scientific research. SDARs are typically linked to using an Internet URL, which have been shown to decay in a time-dependent fashion. What is less clear is whether or not SDAR-producing group size or prior experience in SDAR production correlates with SDAR persistence or whether certain institutions or regions account for a disproportionate number of peer-reviewed resources.MethodsWe first quantified the current availability of over 26,000 unique URLs published in MEDLINE abstracts/titles over the past 20 years, then extracted authorship, institutional and ZIP code data. We estimated which URLs were SDARs by using keyword proximity analysis.ResultsWe identified 23,820 non-archival URLs produced between 1996 and 2013, out of which 11,977 were classified as SDARs. Production of SDARs as measured with the Gini coefficient is more widely distributed among institutions (.62) and ZIP codes (.65) than scientific research in general, which tends to be disproportionately clustered within elite institutions (.91) and ZIPs (.96). An estimated one percent of institutions produced 68% of published research whereas the top 1% only accounted for 16% of SDARs. Some labs produced many SDARs (maximum detected = 64), but 74% of SDAR-producing authors have only published one SDAR. Interestingly, decayed SDARs have significantly fewer average authors (4.33 +/- 3.06), than available SDARs (4.88 +/- 3.59) (p < 8.32 × 10-4). Approximately 3.4% of URLs, as published, contain errors in their entry/format, including DOIs and links to clinical trials registry numbers.ConclusionSDAR production is less dependent upon institutional location and resources, and SDAR online persistence does not seem to be a function of infrastructure or expertise. Yet, SDAR team size correlates positively with SDAR accessibility, suggesting a possible sociological factor involved. While a detectable URL entry error rate of 3.4% is relatively low, it raises the question of whether or not this is a general error rate that extends to additional published entities.

Predictive bioinformatics identifies novel regulators of proliferation in a cancer stem cell model

The cancer stem cell model postulates that tumors are hierarchically organized with a minor population, the cancer stem cells, exhibiting unlimited proliferative potential. These cells give rise to the bulk of tumor cells, which retain a limited ability to divide. Without successful targeting of cancer stem cells, tumor reemergence after therapy is likely. However, identifying target pathways essential for cancer stem cell proliferation has been challenging. Here, using a transcriptional network analysis termed GAMMA, we identified 50 genes whose correlation patterns suggested involvement in cancer stem cell division. Using RNAi depletion, we found that 21 of these target genes showed preferential growth inhibition in a breast cancer stem cell model. More detailed initial analysis of 6 of these genes revealed 4 with clear roles in the fidelity of chromosome segregation. This study reveals the strong predictive potential of transcriptional network analysis in increasing the efficiency of successful identification of novel proliferation dependencies for cancer stem cells.