Aged Microenvironment Research Articles

MicroRNA-31a-5p from aging BMSCs links bone formation and resorption in the aged bone marrow microenvironment.

The alteration of age‐related molecules in the bone marrow microenvironment is one of the driving forces in osteoporosis. These molecules inhibit bone formation and promote bone resorption by regulating osteoblastic and osteoclastic activity, contributing to age‐related bone loss. Here, we observed that the level of microRNA‐31a‐5p (miR‐31a‐5p) was significantly increased in bone marrow stromal cells (BMSCs) from aged rats, and these BMSCs demonstrated increased adipogenesis and aging phenotypes as well as decreased osteogenesis and stemness. We used the gain‐of‐function and knockdown approach to delineate the roles of miR‐31a‐5p in osteogenic differentiation by assessing the decrease of special AT‐rich sequence‐binding protein 2 (SATB2) levels and the aging of BMSCs by regulating the decline of E2F2 and recruiting senescence‐associated heterochromatin foci (SAHF). Notably, expression of miR‐31a‐5p, which promotes osteoclastogenesis and bone resorption, was markedly higher in BMSCs‐derived exosomes from aged rats compared to those from young rats, and suppression of exosomal miR‐31a‐5p inhibited the differentiation and function of osteoclasts, as shown by elevated RhoA activity. Moreover, using antagomiR‐31a‐5p, we observed that, in the bone marrow microenvironment, inhibition of miR‐31a‐5p prevented bone loss and decreased the osteoclastic activity of aged rats. Collectively, our results reveal that miR‐31a‐5p acts as a key modulator in the age‐related bone marrow microenvironment by influencing osteoblastic and osteoclastic differentiation and that it may be a potential therapeutic target for age‐related osteoporosis.

Aging of Hematopoietic Stem Cells Is Driven By Regional Specialization of Marrow Macrophages

While hematopoietic stem cells (HSCs)-intrinsic effects of aging have been explored, less is known about how HSC support is altered by the aged bone marrow microenvironment (BMME). To assess the role of the BMME in HSC aging, we compared the BMME in young (6-12 weeks) and aged (20-24 months) male mice and young (<50 years old; YO) and aged (>50 YO) human volunteers. Aged mice had remodeling of the BMME, with expansion of the marrow cavity and vascular volume compared to young mice. BMME constituents were redistributed within two distinct anatomic regions, namely endosteal bone-associated (BA) and marrow-associated (MA) cells. BA cells in aged mice contained fewer phenotypic mesenchymal/osteoblastic progenitors, with reduction in their ability to constitute colony forming units (CFUs). CFU loss was also observed in aged human volunteers. Aged murine MA had significant expansion of dysfunctional mesenchymal stem cells (MSCs) and activated macrophages (MΦ). Increased MΦ were also detected in aged human marrows.Following this in vivo characterization, we developed an ex vivo co-culture system to determine if aged murine BMME cells could impart aging characteristics to young HSCs. Young murine HSCs co-cultured with aged MA cells acquired phenotypic properties of aged HSCs, including increased CD41+ expression. Single cell RNA sequencing of Long Term-HSCs (LT-HSCs) from young and aged mice also identified upregulation of integrin-β3 (CD61) as a novel marker of aged LT-HSCs. Subsequent flow cytometry analysis confirmed the increase in CD61+ expression in vivo in aged HSCs. Importantly, aged MA - but not BA cells - also increased CD61+ expression in young HSCs ex vivo, highlighting the region-specific remodeling of the BMME that occurs with age.We then used a reductionist approach to identify targetable cellular and molecular regulators of the region-specific BMME-induced HSC aging. CD45+ and Ter119+ depletion in aged MA cells did not induce CD41+ expression in young HSCs, suggesting that a critical BMME component responsible for non-cell-autonomous HSC aging is present within the hematopoietic pool. Since marrow MΦ can regulate HSCs, we co-cultured aged MA MΦ with young MA and found that aged MΦ were sufficient to increase CD41+ expression in young HSCs. The addition of aged MΦ also expanded young MSCs, demonstrating that MΦ orchestrate both BMME remodeling and HSC aging.We next aimed to explore mechanisms by which aged MA MΦ impart aging characteristics to HSCs. Transcriptional analysis of murine MA MΦ demonstrated an increase in inflammatory activation in aged mice compared to young mice. This finding was also present in aged human MΦs. Among the inflammatory signals, interleukin-1β (IL-1β) was identified to be necessary and sufficient to mediate the aging effect of aged MA MΦ on young HSCs. Transcriptional analysis also revealed downregulation of phagocytic programs in aged MA MΦ compared to young MA MΦ. Supporting the transcriptional data, aged MA MΦs cultured in vitro demonstrated impaired ability to engulf senescent neutrophils compared to young MA MΦ. Bone marrow MΦ continuously remove large quantities of senescent neutrophils through phagocytosis, a process also known as efferocytosis. Complementing the in vitro findings, in vivo testing demonstrated that young MA MΦ are primarily responsible for engulfing senescent neutrophils and that aged MA MΦ had reduced engulfment of senescent neutrophils. No phagocytic defect was identified in aged BA MΦ, highlighting the regionalization of MΦ function within the BMME that is differentially impacted with age. Consistent with the systemic impact of the efferocytic defect of aged MA MΦ, aged mice had increased levels of circulating senescent neutrophils and. Moreover, neutrophils from aged mice had increased caspase-1 activity, a signal required for IL-1β activation.Together, these data provide evidence that aging differentially remodels two anatomically distinct BMMEs. Regional specialization of marrow MΦ was differentially impacted by aging and induced aging characteristics in HSCs. We propose that impaired removal of senescent neutrophils by aged MA MΦ increases IL-1β production, leading to local inflammation and disrupted BMME and HSC function in aged mice. Strategies aimed at restoring healthy efferocytic activity as well as diminishing IL-1β production or function could therefore reduce the aging effect on HSCs by rejuvenating the BMME. DisclosuresLiesveld:Onconova: Honoraria; Seattle Genetics: Honoraria.

Endothelial transplantation rejuvenates aged hematopoietic stem cell function.

Age-related changes in the hematopoietic compartment are primarily attributed to cell-intrinsic alterations in hematopoietic stem cells (HSCs); however, the contribution of the aged microenvironment has not been adequately evaluated. Understanding the role of the bone marrow (BM) microenvironment in supporting HSC function may prove to be beneficial in treating age-related functional hematopoietic decline. Here, we determined that aging of endothelial cells (ECs), a critical component of the BM microenvironment, was sufficient to drive hematopoietic aging phenotypes in young HSCs. We used an ex vivo hematopoietic stem and progenitor cell/EC (HSPC/EC) coculture system as well as in vivo EC infusions following myelosuppressive injury in mice to demonstrate that aged ECs impair the repopulating activity of young HSCs and impart a myeloid bias. Conversely, young ECs restored the repopulating capacity of aged HSCs but were unable to reverse the intrinsic myeloid bias. Infusion of young, HSC-supportive BM ECs enhanced hematopoietic recovery following myelosuppressive injury and restored endogenous HSC function in aged mice. Coinfusion of young ECs augmented aged HSC engraftment and enhanced overall survival in lethally irradiated mice by mitigating damage to the BM vascular microenvironment. These data lay the groundwork for the exploration of EC therapies that can serve as adjuvant modalities to enhance HSC engraftment and accelerate hematopoietic recovery in the elderly population following myelosuppressive regimens.

Inhibition of Age-Related Therapy Resistance in Melanoma by Rosiglitazone-Mediated Induction of Klotho.

Purpose: Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein whose serum levels decrease dramatically by age 40. Most studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma.Experimental Design: PPARγ increases klotho levels and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/Wnt5A cross-talk, in vitro and in vivo, and the implications of that for targeted therapy in young versus aged animals.Results: We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF inhibitor-sensitive and BRAF inhibitor-resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors.Conclusions: Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types. Clin Cancer Res; 23(12); 3181-90. ©2017 AACR.

Stem cell therapies in preclinical models of stroke. Is the aged brain microenvironment refractory to cell therapy?

Stem cell therapies in preclinical models of stroke. Is the aged brain microenvironment refractory to cell therapy?

Role of Cell-Intrinsic and Environmental Age-Related Changes in Altered Maintenance of Murine T Cells in Lymphoid Organs

Age-related changes in primary lymphoid organs are well described. Less is known about age-related changes affecting peripheral lymphoid organs, although defects in old peripheral lymph nodes (pLNs) were recently described in both steady state and during viral infection. To address whether such pLN defects were intrinsic to old T cells or extrinsic (due to aging microenvironment), we employed heterochronic parabiosis. We found no age-related intrinsic or extrinsic barriers to T cell circulation and seeding of pLN, spleen, and bone marrow. However, heterochronic parabiosis failed to improve cellularity of old pLN, suggesting an environment-based limit on pLN cellularity. Furthermore, upon parabiosis, pLN of the adult partner exhibited reduced, old-like stromal and T cell cellularity, which was restored following separation of parabionts. Decay measurement of adult and old T cell subsets following separation of heterochronic parabionts delineated both T cell-intrinsic and environmental changes in T cell maintenance. Moreover, parabiotic separation revealed differences between CD4 and CD8 T cell subset maintenance with aging, the basis of which will require further investigation. Reasons for this asymmetric and subset-specific pattern of differential maintenance are discussed in light of possible age-related changes in lymph nodes as the key sites for peripheral T cell maintenance.

Stem cell therapies in preclinical models of stroke. Is the aged brain microenvironment refractory to cell therapy?

Stroke is a devastating disease demanding vigorous search for new therapies. Initial enthusiasm to stimulate restorative processes in the ischemic brain by means of cell-based therapies has meanwhile converted into a more balanced view recognizing impediments that may be related to unfavorable age-associated environments. Recent results using a variety of drug, cell therapy or combination thereof suggest that, (i) treatment with Granulocyte-Colony Stimulating Factor (G-CSF) in aged rats has primarily a beneficial effect on functional outcome most likely via supportive cellular processes such as neurogenesis; (ii) the combination therapy, G-CSF with mesenchymal cells (G-CSF+BM-MSC or G-CSF+BM-MNC) did not further improve behavioral indices, neurogenesis or infarct volume as compared to G-CSF alone in aged animals; (iii) better results with regard to integration of transplanted cells in the aged rat environment have been obtained using iPS of human origin; (iv) mesenchymal cells may be used as drug carriers for the aged post-stroke brains. Conclusion: While the middle aged brain does not seem to impair drug and cell therapies, in a real clinical practice involving older post-stroke patients, successful regenerative therapies would have to be carried out for a much longer time.

A novel Foxn1eGFP/+ mouse model identifies Bmp4-induced maintenance of Foxn1 expression and thymic epithelial progenitor populations.

Although forkhead-box n1 (Foxn1) is a critical thymic epithelial cell regulator in thymus organogenesis, its association with epithelial differentiation and homeostasis in the postnatal and aged thymic microenvironment remains conflicting. Consequently, we have generated a Foxn1eGFP/+ knock-in mouse model that allows for refined investigation of the aging thymic epithelium. This reporter line differs from those previously published in that concomitant expression of enhanced green fluorescent protein enables live cell sorting of Foxn1+ cell populations. Our heterozygotes did not exhibit haploinsufficiency, with Foxn1 expression resembling that of wild-type mice. Comparative analysis between Foxn1 and enhanced green fluorescent protein at both the transcriptional and translational levels revealed co-localization, with progressive down-regulation observed predominantly in the aging cortical epithelium. Supplementation with bone morphogenetic protein (Bmp)-4 enhanced Foxn1 expression and colony forming efficiency in both embryonic and adult progenitor 3D cultures. Strikingly, selective maintenance of immature cortical and medullary epithelial cells was observed which is consistent with the higher Bmp receptor 2 expression levels seen in these progenitor populations. This study demonstrates the significance of our mouse model in unraveling the role of this master regulator in thymus development, homeostasis and aging, providing a faithful reporter system for phenotypic and functional investigations.

Dual Deficiency of mDia1 and Mir-146a in an Age-Related Inflammatory Bone Marrow Microenvironment Induces Ineffective Erythropoiesis in Del(5q) MDS

Myelodysplastic syndromes (MDS) are a group of age-related clonal hematologic diseases characterized by anemia, neutropenia, and thromobocytopenia. The mechanisms of anemia in MDS are unclear, which is partially due to the heterogeneity of MDS involving many cytogenetic and molecular abnormalities. Using a mouse genetic approach, here we show that dual deficiency of mDia1 and miR-146a, two genes located at chromosome 5q that is commonly deleted in MDS, causes an aged-related anemia and ineffective erythropoiesis that closely mimics human MDS. Bone marrow erythropoiesis at various stages was dramatically affected in mDia1/miR-146a double knockout mice, which induced a massive splenomegaly with potent extramedullary erythropoiesis. Old (> 1 year of age), but not young (2-4 months of age), wild type recipient mice that were transplanted with bone marrow cells from mDia1/miR-146a double knockout mice, exhibited severe anemia and rapid lethality, which indicates that the aged microenvironment is important for the development of ineffective erythropoiesis. Consistent with the roles of mDia1 and miR-146a in the innate immune response, the serum levels of TNFα and IL-6 were significantly elevated in mDia1/miR-146a double knockout mice. Pathogen-associated molecular pattern proteins (PAMPs), or damage-associated molecular pattern proteins (DAMPs), whose levels increase in aged microenvironment, both induced TNFα and IL-6 upregulation in mDia1/miR-146a double knockout granulocytes and T cells. Mechanistically, we demonstrated that the anemia and ineffective erythropoiesis was independent of hepcidin expression in mDia1/miR-146a double knockout mice. Instead, pathologic levels of TNFα and IL-6 inhibit erythroid colony formation and differentially affect terminal erythropoiesis through reactive oxygen species-induced caspase-3 activation and cell apoptosis. Our study highlights the dual roles of age-related microenvironment and cytogenetic abnormalities in the pathogenesis of ineffective erythropoiesis in MDS. DisclosuresNo relevant conflicts of interest to declare.

Clonality and Mixed Mutational Signature in Aged Hematopoietic Stem Cells Via Single Cell Variant Analysis

Genomic stability and integrity in Hematopoietic Stem Cells (HSCs) is maintained via DNA damage checkpoints, DNA proofreading and DNA repair (Moehrle et al., 2015; Cell Rep). Despite these mechanisms, recurring and non-recurring mutations accumulate in HSCs upon aging, which correlate with an elevated incidence of myeloproliferative diseases (Rossi, Bryder, and Weissman, Exp. Gerontol. 2007) as well as changes in clonality (Akunuru and Geiger, 2016; Trends in Mol. Med.). The rate at which such mutations accumulate in individual HSCs and the selection advantage/disadvantage that they provide is unclear and is an active area of investigation. Evolutionary theory supports a strong influence of the aged niche on the selection of HSCs clones upon aging (Rozhok, Salstrom, and DeGregori, 2014; Aging). We hypothesized that variant profiling of single HSCs based on RNA transcripts will reveal mutational signatures adapted to the selection pressure of the aging microenvironment.We performed Single cell RNA-seq of daughter cell pairs from young and aged murine HSCs (LSK, CD34-, flk2-). The Genome Analysis Toolkit (GATK; Broad Institute) RNA-seq variant/mutation calling algorithm pipeline was applied with some modifications. Only variants that were observed in both daughter cells of a given pair were selected, which significantly decreased our false discovery rate (tested by a Monte Carlo simulation). First and most interestingly, we observed no significant difference in the overall number of variants/mutations between young and aged HSCs, further supporting our recently published observations on the frequency of DNA mutations in HSCs upon aging (Moehrle et al., 2015; Cell Rep). We then used an approach that takes into account the 3’ and 5’ bases flanking a variant to generate motifs whose frequencies can be mathematically analyzed to deduce characteristic mutational patterns, termed as mutational signatures (Nik-Zainal et al., 2012; Cell). We employed a non-negative matrix factorization (nmf) and principal component analysis (pca) algorithms to generate 10 mutational signatures that explained > 95% of the variance in the dataset. We then analyzed the signatures in a pairwise fashion and selected two signatures with the highest discrimination score between young and aged HSC. Based on this, cells fell into two major groups: group 1 predominantly contained aged single cells (~90% of the cells in this group) whereas, interestingly, group 2 contained a mix of young and aged HSCs. The segregation of young and aged single HSCs counts between groups 1 and 2 was tested using Fisher’s exact test and was statistically significant (p-value 0.0029). These data indicate that while the overall mutational load is not elevated, majority of aged HSCs acquire a mutational signature distinct from young HSCs, while a proportion of aged HSCs present with a young-like HSC signature. Furthermore, our results show that even those cells that have acquired an aging signature aren’t homogeneous and show sub-clustering tendencies, providing the first hint that they may potentially evolve further into more distinct clones. In conclusion, our results show that individual HSCs reflect a mixed mutational profile reminiscent of a non-uniform accumulation of variants. As such signatures are a reflection of underlying mechanisms by which the mutations accumulate (Nik-Zainal et al., 2012; Cell), the proportion of aged HSCs sharing similar mutational signatures but distinct from the young HSCs reveal an aging signature that indicates specific mutational factors and selection pressure of the aging microenvironment. DisclosuresMulaw:NuGEN: Honoraria.

Considerations for successful cancer immunotherapy in aged hosts.

Immunotherapy is now experiencing unprecedented successes in treating various cancers based on new understandings of cancer immunopathogenesis. Nonetheless, although ageing is the biggest risk factor for cancer, the majority of cancer immunotherapy preclinical studies are conducted in young hosts. This review will explore age-related changes in immunity as they relate to cancer immune surveillance, immunopathogenesis and responses to immunotherapy. Although it is recognized that declining T cell function with age poses a great challenge to developing effective age-related cancer immunotherapies, examples of successful approaches to overcome this hurdle have been developed. Further, it is now recognized that immune functions do not simply decline with age, but rather change in ways than can be detrimental. For example, with age, specific immune cell populations with detrimental functions can become predominant (such as cells producing proinflammatory cytokines), suppressive cells can become more numerous or more suppressive (such as myeloid-derived suppressor cells), drugs can affect aged immune cells distinctly and the aged microenvironment is becoming recognized as a significant barrier to address. Key developments in these and other areas will be surveyed as they relate to cancer immunotherapy in aged hosts, and areas in need of more study will be assessed with some speculations for the future. We propose the term 'age-related immune dysfunction' (ARID) as best representative of age-associated changes in immunity.

Friend or foe: the dichotomous impact of T cells on neuro-de/re-generation during aging.

The interaction between T cells and the central nervous system (CNS) in homeostasis and injury has been recognized being both pathogenic (CD4+ T-helper 1 - Th1, Th17 and γδT) and ameliorative (Th2 and regulatory T cells - Tregs). However, in-depth studies aimed to elucidate the precise in the aged microenvironment and the dichotomous role of Tregs have just begun and many aspects remain unclear. This is due, not only to a mutual dependency and reciprocal causation of alterations and diseases between the nervous and T cell immune systems, but also to an inconsistent aging of the two systems, which dynamically changes with CNS injury/recovery and/or aging process. Cellular immune system aging, particularly immunosenescence and T cell aging initiated by thymic involution - sources of chronic inflammation in the elderly (termed inflammaging), potentially induces an acceleration of brain aging and memory loss. In turn, aging of the brain via neuro-endocrine-immune network drives total body systemic aging, including that of the immune system. Therefore, immunotherapeutics including vaccination and “protective autoimmunity” provide promising means to rejuvenate neuro-inflammatory disorders and repair CNS acute injury and chronic neuro-degeneration. We review the current understanding and recent discoveries linking the aging immune system with CNS injury and neuro-degeneration. Additionally, we discuss potential recovery and rejuvenation strategies, focusing on targeting the aging T cell immune system in an effort to alleviate acute brain injury and chronic neuro-degeneration during aging, via the “thymus-inflammaging-neurodegeneration axis”.

Corrigendum: sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance.

Nature 532, 250–254 (2016); doi:10.1038/nature17392 In Fig. 5a of this Letter, the labels PBS and PLX4720 were inadvertently reversed. The corrected Fig. 5a is shown in Fig. 1 of this Corrigendum.

Frizzled with age: an opportunity for ‘gerontological medicine’

Frizzled with age: an opportunity for ‘gerontological medicine’

Stromal senescence establishes an immunosuppressive microenvironment that drives tumorigenesis.

Age is a significant risk factor for the development of cancer. However, the mechanisms that drive age-related increases in cancer remain poorly understood. To determine if senescent stromal cells influence tumorigenesis, we develop a mouse model that mimics the aged skin microenvironment. Using this model, here we find that senescent stromal cells are sufficient to drive localized increases in suppressive myeloid cells that contributed to tumour promotion. Further, we find that the stromal-derived senescence-associated secretory phenotype factor interleukin-6 orchestrates both increases in suppressive myeloid cells and their ability to inhibit anti-tumour T-cell responses. Significantly, in aged, cancer-free individuals, we find similar increases in immune cells that also localize near senescent stromal cells. This work provides evidence that the accumulation of senescent stromal cells is sufficient to establish a tumour-permissive, chronic inflammatory microenvironment that can shelter incipient tumour cells, thus allowing them to proliferate and progress unabated by the immune system.

Kupffer cell subsets differ between young and aged murine livers.

Abstract The immune system, and in particular macrophages, are heavily implicated in wound healing, response to infection, and cancer progression, all of which are linked to mortality in the elderly population. Recently, it has been shown that tissue-resident macrophages from spleen and peritoneum become dysfunctional with aging, likely due to the aged microenvironment. The liver contains approximately 80% of total mammalian tissue-resident macrophages, known as Kupffer cells. Kupffer cells can be divided into two F4/80+ macrophage subsets: CD68+ and CD11b+ cells, which have different origins and functions. Currently, it is unclear how these macrophage subsets are affected by aging. We sought to characterize differences in the Kupffer cell compartment from young (8–10 week) and aged (18–20 month) C57/Bl6 mice using immunofluorescent staining on tissue sections and flow cytometry. Preliminary histological analyses showed that the area of F4/80+ staining did not differ between young and aged mice. Interestingly the area of CD68+ staining was significantly higher in the aged murine livers. In addition, the area of CD32+ staining, which marks both endothelial cells and macrophage precursors, was significantly greater in aged mice, consistent with previous reports of blood vessel thickening in aged livers. Flow cytometry analysis confirmed differences in the macrophage subsets between young and aged murine livers. In addition, functional changes in phagocytosis were noted with aging, suggesting dysfunction of innate abilities. Characterizing the differences in the subsets of tissue-resident macrophages during aging will increase our understanding of age-related pathologies that will aid in the design of therapies in the future.

SFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance.

Cancer is a disease of aging, and aged cancer patients have a poorer prognosis. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumor progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression1–4 we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. We find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signaling cascade in melanoma cells that results in a decrease in β-catenin and MITF, and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to ROS-induced DNA damage, rendering them more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumor progression, offering new paradigms for the design of therapy for the elderly.

Tumor necrosis factor drives increased splenic monopoiesis in old mice.

Aging is accompanied by changes in hematopoiesis and consequently in leukocyte phenotype and function. Although age-related changes in bone marrow hematopoiesis are fairly well documented, changes in extramedullary hematopoiesis are less well described. We observed that 18-22-mo-old mice had larger spleens than young controls and found that the enlargement was caused by increased monopoiesis. Because extramedullary hematopoiesis is often driven by inflammation, we hypothesized that the chronic, low-level inflammation that occurs with age is a causal agent in splenomegaly. To test this theory, we compared the number of monocytes in 18-mo-old tumor necrosis factor-knockout mice, which are protected from age-associated inflammation, and found that they did not have increased extramedullary monopoiesis. To determine whether increased splenic monopoiesis is caused by intrinsic changes in the myeloid precursors that occur with age or by the aging microenvironment, we created heterochronic bone marrow chimeras. Increased splenic monopoiesis occurred in old recipient mice, regardless of the age of the donor mouse, but not in young recipient mice, demonstrating that these cells respond to signals from the microenvironment. These data suggest that decreasing the inflammatory microenvironment with age would be an effective strategy for reducing inflammatory diseases propagated by cells of myeloid lineage, which increase in number with age.

Rejuvenation of Aged Vascular Niches to Enhance Hematopoietic Function

The molecular mechanisms regulating the aging of hematopoietic stem cells (HSCs) are poorly understood. To date, most studies describing age-related alterations have focused on HSC-intrinsic alterations, showing that the absolute number of immunophenotypically defined HSCs increases with age but that aged HSCs exhibit decreased long-term reconstitution potential, self-renewing capacity, altered transciptomes, cell-cycling and responses to cellular stress and DNA damage. Furthermore, old HSCs exhibit a significant myeloid bias at the expense of lymphopoiesis, which is thought to predispose the aging hematopoietic to the development of myeloid neoplasms. While these studies show that cell-intrinsic changes contribute to the aging of the hematopoietic system, most have not adequately addressed the effects of the aging microenvironment. A large body of evidence has demonstrated functional interactions between the HSC and its niche, suggesting that local and systemic factors may regulate HSC function; however, the role of the bone marrow (BM) microenvironment in regulating HSC aging has not been fully elucidated. Understanding the relationship between the BM microenvironment and the HSC during aging may aid in efforts to prevent or reverse the age-related functional decline observed in the hematopoietic system.We have shown that Akt-activated endothelial cells (ECs) within the hematopoietic microenvironment, are indispensable for supporting HSC self-renewal during both steady-state and regenerative hematopoiesis and that EC-specific Mapk signaling drives the differentiation of HSCs into lineage-committed progeny. Here, we demonstrate that young BMECs maintain high levels of Akt signaling, whereas aged ECs exhibit preferential signaling through Mapk. Utilizing a novel HSC/EC co-culture system, we demonstrate that aged BMECs co-cultured with hematopoietic stem and progenitor cells (HSPCs) isolated from young mice inhibit the expansion of repopulating HSCs and are unable to expand aged HSPCs that give rise to long-term, multilineage engraftment. Of note, when we co-cultured aged HSPCs with young BMECs we found that we were able to maintain their functional capacity when assessed by competitive repopulation assays. These data suggest that BMECs play an important role in regulating HSC function.Based on these observations, we set out to test if endothelial Mapk inhibits the vascular niche from supporting functional hematopoiesis. We generated a mouse model in which Mapk was selectively overexpressed in ECs (Mapk VCC) and demonstrated that these mice exhibit a defect in phenotypic and functional HSCs, resembling phenotypes similar to aged HSCs. In particular, transplanted HSCs from Mapk VCC mice lead to diminished engraftment ability with an in increase in myeloid contribution at the expense of B and T cells. To directly test if the functional defects in the HSCs were due to the Mapk-activated vascular niche, we isolated BMECs from these mice and found that Mapk-activated ECs have a decreased ability to support the ex vivo expansion of functional HSCs, with less HSCs in quiescence and more differentiation into granulocytic myeloid cells. Transcriptome and proteomic analyses revealed that aged and Mapk-activated BMECs have similar defects in their pro-HSC angiocrine repertoire, suggesting a possible mechanism for their diminished capacity to instruct and maintain a balanced and healthy hematopoietic system. Furthermore, we utilized an endothelial-based cellular therapy approach to rejuvenate the BM microenvironment and demonstrated that transplantation of young BMECs can enhance hematopoietic recovery and restore HSC function following myeloablative injury in aged mice.Taken together, our in vivo animal model and EC/HSC co-culture system will allow us to screen for angiocrine factors that support the functional attributes of the HSC. Additionally, we have unlocked a potential therapeutic application for the transplantation of ECs following myeloablative treatment. Transplantation of BMECs may create a more permissive microenvironment that promotes an increase in the number of engrafted HSPCs following BM transplantation and accelerates the rate of hematopoietic recovery following radiation or chemotherapeutic regimens decreasing the morbidity/mortality associated with life threatening pancytopenias in the elderly. DisclosuresNo relevant conflicts of interest to declare.

Aging as a driver of leukemogenesis.

Certain leukemia-initiating mutations drive expansion of hemopoietic clones specifically under the selection pressures that prevail in an aged microenvironment.